Bixin and norbixin are natural food colourants synthesised from Bixa orellana L seeds. This study explores the mechanism of apocarotenoids (bixin and norbixin) on the aggressive B16 murine melanoma as an anti-cancer agent. The apocarotenoids inhibited the cell viability of B16 melanoma in a time- and concentration-dependent manner. Apocarotenoids markedly induced cell morphology change, nuclear condensation, DNA fragmentation, and mitochondrial dysfunction through the depletion of glutathione activity, and also induced reactive oxygen species (ROS) generation. Furthermore, apocarotenoids effectively enhanced sub-G1 population and also activated caspase 9 and caspase 3 activities. It decreased and increased the gene expression of the microphthalmia-associated transcription factor, anti-apoptotic Bcl-2 and pro-apoptotic Bax, respectively. Furthermore, molecular docking studies revealed that apocarotenoids inhibited anti-apoptotic protein (Bcl-2 and Bcl-XL) through hydrophobic interaction. These findings suggest that bixin and norbixin act as a promising chemotherapeutic agent against melanoma by inducing apoptosis through mitochondrial-associated death pathway. © 2015 Elsevier Ltd.