It has been reported that genipin, the aglycone of geniposide, induces apoptotic cell death in human hepatoma cells via a NADPH oxidase-reactive oxygen species (ROS)-c-Jun NH(2)-terminal kinase (JNK)-dependent activation of mitochondrial pathway. This continuing work aimed to define that mixed lineage kinase 3 (MLK3) is a key mediator, which connect between ROS and JNK in genipin-induced cell death signaling. In PC3 human prostate cancer cells, genipin stimulated MLK3 activity in concentration- and time-dependent manner. The PC3 cells stably transfected with dominant-negative form of MLK3 was less susceptible to population of the sub-G1 apoptotic cells, activation of caspase, collapse of mitochondrial membrane potential, and release of cytochrome c triggered by genipin, suggesting a crucial role of MLK3 in genipin signaling to apoptotic cell death. Diphenyleneiodonium (DPI), a specific inhibitor of NADPH oxidase, markedly inhibited ROS generation and MLK3 phosphorylation in the genipin-treated cells. Pretreatment with SP0600125, a specific inhibitor of JNK but neither U0126, a specific inhibitor of MEK1/2 nor PD169316, a specific inhibitor of p38 suppressed genipin-induced apoptotic cell death. Notably, both the phosphorylation of JNK and induction of c-Jun induced by genipin were markedly inhibited in PC3-EGFP-MLK3 (K144R) cells expressing a dominant-negative MLK3 mutant. Taken together, our observations suggest genipin signaling to apoptosis of PC3 cells is mediated via activation of ROS-dependent MLK3, which leads to downstream activation of JNK.