The expression of c-myc promotes cell proliferation and also sensitizes cells to various extracellular apoptotic stimuli. However, signal pathways regulating the function of Myc proteins during apoptosis are unknown. c-Jun N-terminal kinase (JNK) is activated by various apoptotic stimuli, but neither the target molecule(s) or the action of JNK has been identified in Myc-mediated apoptosis. Here, we found that JNK selectively interacted with, and phosphorylated, c-Myc at Ser-62 and Ser-71 as confirmed with phospho-c-Myc-specific antibodies. Interestingly, dominant negative mutant JNK(APF) impaired the c-Myc-dependent apoptosis, but not mutated c-Myc (S62A/S71A)-dependent apoptosis triggered by UV irradiation. Furthermore, c-Myc (S62A/S71A)-expressing NIH3T3 cells were not sensitized like wild type c-Myc-expressing NIH3T3 cells to JNK-activating apoptotic stimuli, such as UV and Taxol. These results indicate that the JNK pathway is selectively involved in the c-Myc-mediated apoptosis and that the apoptotic function of c-Myc is directly regulated by JNK pathway through phosphorylation at Ser-62 and Ser-71.