Gregory is the manager of Gregory House, a mysterious hotel that exists in an alternate dimension and functions as a purgatorial space for lost souls seeking to escape reality. He appears as an elderly anthropomorphic mouse with grey fur, a creased forehead, visible bags under his eyes, and a few warts on his face, with distinctive purple irises. His flat, shoulder-length hair is a faded blonde color, and he typically wears a pink trench coat over a black and red horizontally striped shirt. He is often seen carrying a brass candle holder with a wax candle or a set of keys.

His personality merges superficial politeness with psychological manipulation. He has a habit of appearing without warning, murmuring cryptic or troubling observations, and chuckling, often when he is being deceptive. He addresses male guests as my friend and female guests as my dear, all while expertly exploiting their emotional and mental vulnerabilities to unravel their sanity. Gregory's core motivation is the collection of souls, which he gathers not for himself but for his mother, Gregory Mama. He deeply fears her, a fear that compels his compliance despite his outward resentment and occasional contemplation of abandoning or harming her. It is implied that he is a manifestation of suppressed human desires and dreams, specifically the longing to escape a cold and boring reality for something more alluring.

Within his own dimension, Gregory wields near-absolute control. He can appear and disappear unpredictably to stalk guests through the hotel's halls. However, he cannot permanently leave Gregory House; any attempt to flee his reality ultimately fails. This theme is central to his role in The Last Train, where Gregory himself becomes a protagonist seeking to escape his own existence. He boards a bizarre train populated by residents from his hotel and other strange entities, hoping to break free from his fate and inner demons. During the journey, he offers seemingly encouraging advice to others, such as two Dead Bodies and a pair of Trap Mice, about overcoming their limitations. He demonstrates his dark, voyeuristic nature by observing with amusement as their attempts end in disaster, such as the Trap Mice destroying each other when they can no longer fight their instincts. Ultimately, the train is revealed to be a toy, and his escape attempt fails, forcing his inevitable return to Gregory House.

Gregory has several key relationships. He is often accompanied by his young, mischievous grandson James, who delights in playing pranks and exposing Gregory's secrets, such as his hidden collection of pornographic magazines. His relationship with his mother is one of fear and resentment, and she frequently abuses him physically, such as rapping him on the skull with her staff. He interacts with a vast array of other bizarre residents, including the bloodthirsty nurse Catherine, the spectral Neko Zombie whom he imprisoned, and the judge of souls known as Judgement Boy. In his interactions with guests, such as the woman known as the Second Guest, he adopts a faux-paternal guise to intensify their negative emotions, though his manipulations are sometimes exposed by James, leading to rebellion against him.

While he does not typically develop in a traditional sense, his attempt to escape in The Last Train highlights his own entrapment within the cycle of his purgatorial existence. He is apparently immortal and nigh-invulnerable, having survived events such as being struck in the head with an axe and the burning of his hotel. The only way to potentially destroy him is to destroy the thoughts and feelings tied to his world. He demonstrates the notable ability to break the fourth wall, maintaining narrative awareness and addressing viewers directly in his closing monologues, reinforcing the inescapable nature of his realm and assuring guests that their rooms remain reserved for eternity.