Hepatocellular carcinoma (HCC) is the most common liver cancer and is known to be resistant to conventional chemotherapy. The use of herbal medicine and supplements has increased over recent decades following side effects and resistant to conventional chemotherapy. The seeds of Bixa orellana L. commonly known as annatto have recently gained scientific attention due to presence of a carotenoid bixin for its substantial anticancer properties. However, molecular mechanisms underlying bixin-induced apoptosis are still unclear. Treatment of bixin significantly decreased the number of Hep3B cells and morphological study revealed the change in cellular and nuclear morphology that trigger the events of apoptosis confirmed by annexin V/PI staining. Further DCFDA and rhodamine 123 spectrofluorimetry study showed elevation in reactive oxygen species (ROS) production and loss of mitochondrial membrane potential (MMP), respectively. ROS production caused DNA damage and apoptosis was marked by cell cycle arrest, up-regulation of Bax and FasL protein as well as cleavage of caspase-9, caspase-8 and caspase-3 protein. Docking study with pro-apoptotic molecule Bax and surface Fas ligand exhibited energetically favourable binding interaction. Collectively, these results suggest that bixin capable of modulating the extrinsic and intrinsic molecules of apoptosis indicating its potential for development of promising candidate for hepatocellular carcinoma.