HMGB1 belongs to the high mobility group (HMG) protein family, characterized by the presence of structurally conserved DNA-binding HMGB domain. In addition to two DNA-binding domains connected by a short linker, HMGB1 contains a short N-terminal sequence and an intrinsically disordered C-terminal region. The negatively charged C-terminal HMGB1 domain modulates both DNA–protein and protein–protein interactions. HMGB1 is involved in nearly all major cellular processes, including DNA repair and transcription, and contributes to chromatin organization. Moreover, HMGB1 functions as a damage-associated molecular pattern that initiates inflammatory responses. Given its essential role in maintaining normal cellular functioning, dysregulation of HMGB1 activity is associated with various pathologies, including cancer, cardiovascular diseases, inflammatory conditions, and autoimmune disorders. Therefore, to maintain normal functions, precise regulation of HMGB1 activity is critical and occurs at the levels of gene expression, posttranslational modification, and protein stability. Recent studies have identified specific E3 ubiquitin ligases that promote HMGB1 degradation via the ubiquitin-proteasome pathway. This review summarizes the current knowledge of these enzymes and discusses their functional roles.