Proteasome inhibitor PS-341 induces growth arrest and apoptosis of multiple myeloma (MM) cells via inactivation of NF-κB in vitro and has afforded some objective responses in individuals with relapsed, refractory MM. However, the activity of PS-341 against non-hematological malignancies remains to be fully elucidated. In this study, we found that PS-341 induced growth arrest and apoptosis of NCI-H520 and -H460 non-small cell lung cancer (NSCLC) cells in conjunction with markedly up-regulated levels of p21waf1 and p53, and down-regulation of bcl-2 protein in these cells. Also, PS-341 caused phosphorylation of c-Jun NH2-terminal kinase (JNK) and c-Jun, and enhanced AP-1/DNA binding activities in these cells as measured by western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Interestingly, when the JNK/ c-Jun/AP-1 signal pathway was disrupted by the JNK inhibitor SP600125, the ability of PS-341 to inhibit the growth of NSCLC cells and to up-regulate the levels of p21waf1 in these cells was blunted, but the expression of p53 was sustained at a high level, suggesting that the JNK/c-Jun/AP-1 signal pathway might mediate the anti-lung cancer effects of PS-341, with p21waf1 playing the central role. Thus, PS-341 might be useful for the treatment of individuals with NSCLC. (Cancer Sci 2004; 95: 176–180)