The cyclin-dependent kinase inhibitor p21^Cip1 regulates multiple cellular functions and protects cells from genotoxic and other cellular stresses. Activation of apoptosis signal-regulating kinase 1 (ASK1) induced by inhibition of mTOR signaling leads to sustained phospho-c-Jun that is suppressed in cells with functional p53 or by forced expression of p21^Cip1. Here we show that small deletions of p21^Cip1 around S98 abrogate its association with ASK1 but do not affect binding to Cdk1, hence distinguishing between the cell cycle-regulating functions of p21^Cip1 and its ability to suppress activation of the ASK1/Jun N-terminal protein kinase (JNK) pathway. p21^Cip1 is phosphorylated in vitro by both ASK1 and JNK1 at S98. In vivo phosphorylation of p21^Cip1, predominantly carried out by ASK1, is associated with binding to ASK1 and inactivation of ASK1 kinase function. Binding of p21^Cip1 to ASK1 requires ASK1 kinase function and may involve phosphorylation of S98.