Hint1 is a member of the evolutionarily conserved family of histidine triad proteins that acts as a haplo-insufficient tumor suppressor inducing spontaneous tumor formation in Hint(+/-) and Hint(-/-) mouse models. However, the molecular mechanisms for the tumor-suppressing activity are poorly defined. In this respect, we have recently shown that Hint1, by interaction with Pontin and Reptin, inhibits T-cell factor/beta-catenin-mediated transcription of Wnt target genes. In this study, we have found that, after transient transfection with Hint1, SW480 and MCF-7 cells undergo apoptosis as analyzed by pro-caspase-3 and poly(ADP-ribose) polymerase cleavage, M30 CytoDEATH staining, cytochrome c release, and DNA fragmentation enzyme-linked immunosorbent assay. Hint1 is involved in the regulation of apoptotic pathways by inducing an up-regulation of p53 expression coinciding with an up-regulation of the proapoptotic factor Bax and a concomitant down-regulation of the apoptosis inhibitor Bcl-2. Bad and Puma levels remained unchanged. Further analyses revealed that Hint1 is associated with the Bax promoter and is a component of the Tip60 histone acetyltransferase complex and, in this context, appears to be involved in the regulation of Bax expression. Knockdown of Hint1 by short hairpin RNA resulted in down-regulation of p53 and Bax but had no effect on Bcl-2 expression. A mutant Hint1 (H112N) protein defective in enzymatic activity as an AMP-NH(2) hydrolase was not impaired in induction of apoptosis, suggesting that the Hint1 pro-apoptotic activity is independent of the Hint1 enzymatic activity.