High-mobility group box 1 protein (HMGB1) is a nonhistone chromosomal protein discovered more than 30 years ago. It is an abundant nuclear protein that has a dual function-in the nucleus, it binds DNA and participates in practically all DNA-dependent processes serving as an architectural factor. Outside the cell, HMGB1 plays a different role-it acts as an alarmine that activates a large number of HMGB1-"competent" cells and mediates a broad range of physiological and pathological responses. This universality makes it an attractive target for innovative therapeutic strategies in the treatment of various diseases. Here we present an overview of the major nuclear and extracellular properties of HMGB1 and describe its interaction with different molecular partners as specific receptors or inhibitors, which are important for its role as a target in multiple diseases. We highlight its pivotal role as a target for cancer treatment at two aspects: first in terms of its substantial impact on the repair capacity of cancer cells, thus affecting the effectiveness of chemotherapy with the antitumor drug cis-platinum and, second, the possibility to be targeted by microRNAs influencing different pathways of human diseases, thus making it a promising candidate for a new strategy for therapeutic interventions against various pathological conditions but mainly cancer.