The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis. Previous studies by our group showed that CTN triggers apoptosis in mouse embryonic stem cells, as well as embryonic developmental injury. Here, we investigated the precise mechanisms governing this apoptotic effect in osteoblasts. CTN induced apoptotic biochemical changes in a human osteoblast cell line, including activation of c-Jun N-terminal kinase (JNK), loss of mitochondrial membrane potential, and caspase-3 and p21-activated protein kinase 2 (PAK2) activation. Experiments using a JNK-specific inhibitor, SP600125, and antisense oligonucleotides against JNK reduced CTN-induced activation of both JNK and caspase-3 in osteoblasts, indicating that JNK is required for caspase activation in this apoptotic pathway. Experiments using caspase-3 inhibitors and antisense oligonucleotides against PAK2 revealed that active caspase-3 is essential for PAK2 activation. Moreover, both caspase-3 and PAK2 require activation for CTN-induced apoptosis of osteoblasts. Interestingly, CTN stimulates two-stage activation of JNK in human osteoblasts. Early-stage JNK activation is solely ROS-dependent, whereas late-stage activation is dependent on ROS-mediated caspase activity, and regulated by caspase-induced activation of PAK2. On the basis of these results, we propose a signaling cascade model for CTN-induced apoptosis in human osteoblasts involving ROS, JNK, caspases, and PAK2. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.