High-Mobility Group Box 1 (HMGB1) is an abundant, highly conserved, non-histone nuclear protein present in almost all eukaryotic cells. In inflammatory conditions, HMGB1 is actively secreted from immune cells in the extracellular matrix, where it behaves as a proinflammatory cytokine. Once released, it can bind to cell-surface receptors, such as the Receptor for Advanced Glycation End products (RAGE) and Toll-Like Receptors (TLR) 2, 4 and 9, and mediate various cellular responses, including the induction of cell migration/proliferation and the release of other proinflammatory cytokines. Moreover, HMGB1 is able to contribute to the pathogenesis of various chronic inflammatory and autoimmune diseases as well as of cancer. Given the multiple roles of HMGB1 in these pathologies, identification of inhibitors of this protein is of considerable clinical interest. We here identified novel G-quadruplex (G4) forming aptamers as potential HMGB1 inhibitors. Using SELEX technology, we selected 14 G4-forming DNA sequences from a properly designed G-rich oligonucleotide library. These aptamers have been fully characterized in a biologically relevant buffer using several biophysical techniques to determine their preferred conformation as well as their thermal and enzymatic stability. Moreover, we evaluated the interaction between these aptamers and HMGB1, as well as their ability to inhibit HMGB1-induced migration in cancer cells so to identify the best candidates for future in vivo assays aimed at repressing the pathological functions induced by the target protein.