Charlie is a 32-year-old man living in New York City with an intellectual disability and an immense drive to learn. He works as a janitor at Mr. Donner's bakery and attends night classes at the Beekman College Center. Selected for a groundbreaking experimental surgery designed to increase his intelligence, he documents his experiences in a series of progress reports. He is earnest and highly motivated, carrying a deep desire to form meaningful connections with others and understand the world around him.

Alice is Charlie's teacher at the Beekman College Center for adults with intellectual disabilities. She recognizes Charlie's exceptional drive early on and remains a steady source of support throughout his rapid transformation. She possesses a strong sense of fairness and cares deeply for Charlie, though she often worries about the psychological toll the experiment might take on him.

Algernon is a white laboratory mouse and the first successful animal subject of the intelligence-enhancing experimental procedure. He displays remarkable cognitive abilities, running complex mazes and solving puzzles with ease. He serves as both a literal and symbolic counterpart to Charlie's own intellectual journey.

Professor Nemur is a senior researcher and co-director of the experiment at the Beekman lab. He is highly driven by the desire for scientific success and academic recognition, sometimes prioritizing the research itself over his subjects' emotional needs. He operates under considerable pressure from his well-connected wife to achieve a major scientific breakthrough.

Dr. Strauss is a psychiatrist and neurosurgeon working alongside Professor Nemur on the intelligence experiment. He is responsible for conducting Charlie's therapy sessions and monitoring his psychological well-being. Unlike Nemur, Strauss is more attuned to the emotional and social challenges Charlie faces as his intellect begins to rapidly outpace his emotional maturity.

Burt is a graduate student working under Professor Nemur and Dr. Strauss at the Beekman lab. He runs the day-to-day psychological and cognitive tests on both Charlie and Algernon. He provides a calm, reasonable presence in the laboratory environment, often helping Charlie understand the complex motivations of the senior scientists.

Fay is Charlie's free-spirited, eccentric neighbor who works as an artist. She loves dancing, drinking, and living without strict rules or societal expectations. Her vivacious and unconventional lifestyle offers Charlie an entirely new way of experiencing the world outside the rigid confines of the scientific laboratory.

Mr. Donner is the owner of the bakery where Charlie works and a long-time friend of Charlie's Uncle Herman. He took Charlie in when his family abandoned him, providing him with a job and stability. Though he cares for Charlie, he is also heavily influenced by the attitudes of his other employees and the social dynamics of the bakery floor.

Gimpy is a senior baker at Mr. Donner's shop. He occasionally attempts to teach Charlie new skills, sometimes rewarding him with small trinkets, though he also partakes in the casual mockery of Charlie's disability. His actions present Charlie with early ethical dilemmas as Charlie's intelligence grows.

Joe is a worker at Donner's Bakery whom Charlie initially considers a close friend. Joe frequently makes Charlie the butt of his jokes, getting him drunk and abandoning him for amusement, taking advantage of Charlie's inability to understand when he is being mocked.

Frank is another employee at Donner's Bakery who joins Joe Carp in playing cruel pranks on Charlie. He views Charlie's intellectual disability as a source of workplace entertainment, organizing outings specifically to humiliate him.

Fanny is an employee at Donner's Bakery who is generally kinder to Charlie than the male workers. She is the one who originally suggested that Charlie attend the night classes at the Beekman College Center, showing a genuine interest in his self-improvement.

Rose is Charlie's mother, whose deep shame over his intellectual disability heavily influenced his early life. In Charlie's surfacing memories, she alternates between harsh rejection and an obsessive, desperate drive to make him "normal" before eventually forcing him out of the family home entirely.

Matt is Charlie's father, who eventually opens his own barbershop in the Bronx. In Charlie's memories, Matt is portrayed as more accepting of Charlie's disability than Rose, attempting to defend him against her demands, though he ultimately fails to prevent Charlie's exile from the family.

Norma is Charlie's younger sister. As a child, she resented Charlie's intellectual disability and the tension it brought to their household, feeling frustrated that her brother was not "normal." Her presence in the family dynamic deeply influenced how their mother treated Charlie.

Uncle Herman is Charlie's relative who took custody of him after his parents removed him from their home. He set up Charlie's job at Donner's Bakery, ensuring his nephew would have a safe place to work and live before eventually passing away.

Dr. Guarino is a specialist Charlie's parents took him to as a child, hoping for a cure for his intellectual disability. Although his medical treatments were ineffective, Charlie remembers him fondly because he was one of the few people who treated him with genuine human dignity.