Abstract: Background: Newcastle Disease Virus (NDV) is a negative-sense single stranded RNA virus that causes a Newcastle Disease (ND), a contagious disease of domestic poultry and wild birds characterized by gastro-intestinal, respiratory and nervous signs. Despite the negative effects of NDV to avian species, this virus was reported to possess significant oncolytic activity against mammalian cancerous cells. Methodology: In this study, the antiproliferative and apoptotic effect of NDV strain AF2240 on human promyelocytic leukaemia HL60 cell line were assessed using MTT proliferation assay, microscopic observation, DNA fragmentation, annexin V-FITC assay, caspase-3/7, 8, 9 assays and caspase-3/7, 8, 9 inhibition assays. Results: The proliferation of HL60 cells was inhibited when treated with cytotoxic titers (CD25, CD50 and CD75) of NDV AF2240 for a period of 72 h. Result from microscopic observation showed NDV AF2240 caused inhibition of cell growth and the treated cells exhibited morphological features of apoptosis and a ladder-like pattern of DNA, which is a hallmark of apoptosis. The proportion of cells in early and late apoptosis was quantified by using annexin V-FITC staining and analysed with flow cytometer. The percentage of cells in early apoptosis after treatment with NDV AF2240 at CD50 titer for 24 and 48 h were 16.27±0.25 and 25.93±1.2%, respectively. Late-apoptotic cells were increased from 3.15±0.07 and 6.85±1.05%, respectively. The mechanism of apoptosis through activation of caspases induced by NDV AF2240 was also analysed. The results suggested that apoptosis in NDV-infected tumor cells is dependent on caspase as both iniatiator caspases; caspase 8 and 9 were activated. Activation of caspase-3/7 were also detected in the cells treated with NDV AF2240. Furthermore, apoptosis by NDV AF2240 was effectively inhibited by ZVAD-FMK indicate that NDV AF2240-induced apoptosis is entirely dependent on caspase activation. Conclusion: To conclude, NDV AF2240 was found to have antiproliferative and apoptotic effects on HL60 cells. It has been shown from this study that NDV AF2240 infection resulted in the activation of both intrinsic and extrinsic apoptotic pathways.