ING (inhibitor of growth) tumor suppressors regulate cell-cycle checkpoints, apoptosis, and ultimately tumor suppression. Among the ING family members, p33(ING1b) is the most intensively studied and plays an important role in the cellular stress response to DNA damage. Here we demonstrate that there is basal phosphorylation of p33(ING1b) at Ser-126 in normal physiological conditions and that this phosphorylation is increased on DNA damage. The mutation of Ser-126 to alanine dramatically shortened the half-life of p33(ING1b). Furthermore, we found that both Chk1 and Cdk1 can phosphorylate this residue. Interestingly, while Cdk1 can phosphorylate p33(ING1b) at Ser-126 in nonstress conditions, Chk1 predominantly phosphorylates this residue on DNA damage, which suggests that p33(ING1b) is a downstream target of the ATM/ATR response cascade to genotoxic stress. More importantly, our data indicate that the Ser-126 residue plays a key role in regulating the expression of cyclin B1 and proliferation of melanoma cells.