Apoptosis signal-regulating kinase 1 (ASK1) is a serine/threonine kinase that mediates cell stress signaling initiated by diverse stimuli, such as H2O2 and TNFα. Owing to its critical role in promoting apoptosis, ASK1 activity is highly controlled in cells. Phosphorylation of ASK1 at Thr-845 has been correlated with its activation, while phosphorylation at Ser-967 negatively controls its death promoting activity. Here, we report the identification of a novel phosphorylation site at Ser-1034 in the C-terminal regulatory domain of ASK1. Mutating Ser-1034 to an unphosphorylatable Ala led to increased catalytic activity of ASK1 and enhanced proapoptotic function of ASK1. Thus, the proapoptotic function of ASK1 is suppressed in part by phosphorylation at its C-terminal regulatory domain, which may couple upstream survival kinases to the death regulatory machinery.