The c-Jun amino-terminal kinase (JNK) is an important player in inflammation, proliferation, and apoptosis. Here, by using a yeast two-hybrid technology, p65 subunit of NF-κB transcription factor was identified as a partner of JNK3. We show that JNK3 physically associated with p65 in vivo and in vitro. Overexpression of JNK3 inhibited NF-κB- dependent transcription induced by TNFα. It was demonstrated that JNK3 decreased NF-κB binding to its cognate DNA sequences and NF-κB target genes expression. Taken together, these data suggest that JNK3 may function in vivo as a modulator in suppressing the transcriptional activity of p65.