Background: Arsenic trioxide and 1,25-(OH)2D3 (vitamin D3) are used for the treatment of lymphocytic leukemia. However, the effects of combined treatment of these drugs are controversial. In this study, the combined effects of these drugs on the induction of apoptosis in NB4 cells were investigated using the neutral comet assay. Methods: NB4 cells were treated with various doses of arsenic trioxide (0.1 – 3 μM) and vitamin D3 (100 – 600 nM (alone or in combination. Twenty-four hours after treatment, neutral comet assay was performed and apoptotic cells were scored under a fluorescent microscope following staining with ethidium bromide. Results: Results show that all doses of arsenic trioxide used in this study induced apoptosis in NB4 cells. The frequency of induced apoptosis was dose dependent and significantly higher than the controls (P<0.05 – 0.01). In contrast, vitamin D3 at concentrations of 100 – 600 nM produced no significant effect on apoptosis induction compared to the controls. Treatment of NB4 cells with a combination of arsenic trioxide and vitamin D3 resulted in reduction of apoptosis induced by arsenic trioxide which was not dependent on the dose of vitamin D3 (P<0.05). Conclusion: Results indicate that arsenic trioxide is a potent inducer of apoptosis in NB4 cells and vitamin D3 significantly decreased the sensitivity of cells to the induction of apoptosis by arsenic trioxide. These findings suggest that 1,25-(OH)2D3 might be involved in anti-apoptotic processes via reactive oxygen species scavenging or other mechanisms not yet known.