Metastatic cancers including melanoma are frequently associated with increased resistance to apoptosis induced by various therapeutic modalities, and the success of systemic therapy for the treatment of metastatic melanoma is minimal. In the present study, we demonstrated the ability of apoptosis-related protein (APR)-2 to trigger cell death via mechanism mediated by both endoplasmic reticulum (ER) stress [as evidenced by the increase of intracellular Ca(2+) release, the activation of both, inositol-requiring enzyme 1α (IRE1α) and calpain and cleavage of caspase-4] and mitochondrial dysregulation as evidenced by the loss of mitochondrial membrane potential, Cytochrome c release and cleavage of caspases-9 and -3, and poly adenosine diphosphate ribose polymerase (PARP). Also, the activation of apoptosis signal-regulating kinase (ASK) 1, c-jun-N-terminal kinase (JNK) and the transcription factors AP-1 and p53, and the induction of Bax expression were noted in APR-2-expressing cells. Both immune fluorescence staining and western blotting revealed the localization of APR-2 at ER and Bax protein at both mitochondria and ER. However, data of inhibitory experiments demonstrated that APR-2-induced apoptosis of melanoma cells is mediated by three parallel pathways: one of them IRE1/tumour necrosis factor receptor-associated factor 2/ASK1/JNK/Cyt.c/caspase-9/caspase-3/PARP) seems to be mitochondrial dependent, whereas, the other two pathways namely calpain/caspase-4/caspase-9/caspase-3/PARP and protein kinase RNA-like ER kinase/ATF4/C/EBP homologous protein (CHOP)/Bim seem to be mitochondrial independent. In conclusion, our data provide insight into the molecular mechanism of APR-2-induced apoptosis and suggest APR-2 gene transfer as an alternative approach for the treatment of chemoresistance melanoma metastasis.