<p>Mono-ubiquitination of histone H2A at lysine 119 (H2AK119Ub) is deposited by the Polycomb repressive complex 1 (PRC1) and represents an abundant post-translational modification (PTM) of histones. H2AK119Ub is crucially involved in the regulation of a wide range of biological processes, including organization of the genome into distinct functional domains, gene silencing, and the maintenance of cell identities during development, among others. Biochemically, the deposition and removal of H2AK119Ub are tightly controlled in cells owing to a dynamic balance between the specific “writers” (i.e., PRC1) and “erasers” (i.e., deubiquitinases (DUBs) such as BAP1 and USP16). Furthermore, the increasing evidence establishes a notion that H2AK119Ub serves as a signal for recruiting specific “readers” (such as JARID2, DNMT3A, RYBP, SSX, and RSF1), which elicit the critical downstream effects such as modulating gene transcription, maintaining genome integrity, and shaping cell identity. This H2AK119Ub-based signaling is often perturbed in human diseases, pointing to a connection between its dysregulation and pathological development. This review is aimed at providing a timely, in-depth analysis of the molecular machinery governing H2AK119Ub, its interactions with other chromatin factors, and its causal role in the onset and progression of diseases, notably cancer.</p>