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. -- Garate, M., Campos, E. I., Bush, J. A., Xiao, H., Li, G. Phosphorylation of the tumor suppressor p33^ING1b at Ser-126 influences its protein stability and proliferation of melanoma cells.