p53 function is modulated by several covalent and non-covalent modifiers. The architectural DNA- binding protein, High Mobility Group protein B-1 is a unique activator of p53. HMGB-1 protein is structured into two HMG-box domains, namely A-box and B-box, connected to a long highly acidic C-terminal domain. Here we report that both the C-terminal domain and A-box of HMGB-1 are critical for stimulation of p53-mediated DNA binding to its cognate site. Though deletion of these domains showed minimal effect in activation of p53-mediated transcription from the DNA template as compared to full-length HMGB-1, truncation of both the domains indeed showed significant reduction of transcriptional activation from the chromatin template as observed in DNA binding. Using transient transfection assays we showed that the C-terminal acidic domain and A-box of HMGB-1 are critical for the enhancement of the p53-mediated transactivation in vivo. Furthermore, the C-terminal domain and A-box deleted HMGB-1 could not activate p53-dependent apoptosis above the basal level. In conclusion, these results elucidate the role of acidic C-terminal domain and A-box of HMGB-1 in p53-mediated transcriptional activation and its further downstream effect.