The notorious resistance of melanoma cells to drug treatment can be overcome by expression of a 50-aa peptide derived from activating transcription factor 2 (ATF2(50-100)). Here we demonstrate that ATF2(50-100) induced apoptosis by sequestering ATF2 to the cytoplasm, thereby inhibiting its transcriptional activities. Furthermore, ATF2(50-100) binds to c-Jun N-terminal kinase (JNK) and increases its activity. Mutation within ATF2(50-100) that impairs association with JNK and the inhibition of JNK or c-Jun expression by RNA interference (RNAi) reduces the degree of ATF2(50-100)-induced apoptosis. In contrast, TAM67, a dominant negative of the Jun family of transcription factors, or JunD RNAi attenuates sensitization of melanoma cells expressing ATF2(50-100) to apoptosis after treatment with anisomycin, which is used as a model drug. Mutations within the JNK binding region of ATF2(50-100) or expression of TAM67 or JunD RNAi attenuates inhibition of melanoma's tumorigenicity by ATF2(50-100). We conclude that inhibition of ATF2 in concert with increased JNK/Jun and JunD activities is central for the sensitization of melanoma cells to apoptosis and inhibition of their tumorigenicity.