HMGB1 is one of the key proteins of the cell. HMGB1 performs its main functions predominantly in the cell nucleus, as an essential component of DNA–protein and multiprotein complexes. It plays an important role in various cellular processes, such as transcription, replication, and DNA repair. However, it has also been found outside the nucleus: in the cytoplasmic and extracellular space. Despite numerous publications on the structure and functioning of HMGB1, the molecular mechanisms that underlie the vast variety of functions performed by this protein still remain unclear. In this paper, we report recent data on the organization of the protein structure and its effects on HMGB1 interactions with DNA and other proteins.