The present study was designed to assess the mechanism of N-phenethyl-2-phenylacetamide (NPPA), one of three new compounds isolated from Xenorhabdus nematophilus, on the induction of apoptosis in U937 cells. NPPA displayed strong inhibitory effects on cell proliferation and viability of U937 cells and induced apoptosis. Investigation of the mechanism of NPPA-induced apoptosis revealed that treatment with NPPA produced morphological features of apoptosis and DNA fragmentation. This was associated with caspase-3 activation and cleavage of poly(ADP-ribose) polymerase. U937 cells treated with NPPA demonstrated cytochrome c accumulation in the cytosol during apoptosis induction. Pretreatment of cells with the pan-caspase inhibitor (z-VAD-fmk) prevented NPPA-induced apoptosis. These results suggested that NPPA induces apoptosis through cytochrome c-dependent caspase-3 activation in U937 cells. In late stage of apoptosis, 18 kDa fragment of Bax was generated with the down-regulation of the expressions of XIAP following NPPA treatment, suggesting that the modulation of Bax and XIAP proteins plays some roles in NPPA-mediated apoptosis. Pretreatments of z-VAD-fmk and the calpain inhibitor, calpeptin, inhibited Bax cleavage. Pretreatment of z-VAD-fmk restored the expression level of XIAP, but pretreatment of calpeptin did not. These results suggest that the elevated caspase activities cleave XIAP in this experiment. And Bcl-2 over-expression attenuates NPPA-induced apoptosis by inhibiting caspase-3 activation, and subsequently inhibits calpain autolysis and Bax cleavage. These results suggested that Bax cleavage is mediated by calpain, and calpain activation may be caspase-dependent. Taken together, the apoptotic effects of NPPA may be related, in part to the caspase-3 activation, the down-regulation of XIAP, and Bax cleavage mediated by caspase-dependent calpain activation.