High mobility group box protein 1 (HMGB1) is a highly versatile, abundant, and ubiquitously expressed, non-histone chromosomal protein, which belongs to the HMGB family of proteins. These proteins form an integral part of the architectural protein repertoire to support chromatin structure in the nucleus. In the nucleus, the role of HMGB1 is attributed to its ability to bind to undamaged DNA, damaged DNA, and alternative (i.e. non-B) DNA structures with high affinity and subsequently induce bending of the DNA substrates. Due to its binding to DNA, HMGB1 has been implicated in critical biological processes, such as DNA transcription, replication, repair, and recombination. In addition to its intracellular functions, HMGB1 can also be released in the extracellular space where it elicits immunological responses. HMGB1 associates with many different molecules, including DNA, RNA, proteins, and lipopolysaccharides to modulate a variety of processes in both DNA metabolism and in innate immunity. In this review, we will focus on the implications of the interactions of HMGB1 with nucleic acids in DNA repair and immune responses. We report on the roles of HMGB1 in nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR) and DNA double-strand break repair (DSBR). We also report on its roles in immune responses via its potential effects on antigen receptor diversity generation [V(D)J recombination] and interactions with foreign and self-nucleic acids. HMGB1 expression is altered in a variety of cancers and immunological disorders. However, due to the diversity and complexity of the biological processes influenced by HMGB1 (and its family members), a detailed understanding of the intracellular and extracellular roles of HMGB1 in DNA damage repair and immune responses is warranted to ensure the development of effective HMGB1-related therapies.