In developed countries, cancer is the leading cause of death among all populations, particularly the elderly. Vitamin D has anti-proliferative, pro-apoptotic, and anti-angiogenic effects on cells, which has strengthened its position as a cancer preventative agent. Inecalcitol is the14-epi-analogue of calcitriol (1,25(OH)2-vitamin D3), which inhibits cancer cell proliferation more effectively than calcitriol. This study was conducted to evaluate the antiproliferative effect of Inecalcitol and the synergistic effect of Inecalcitol in combination with the anti-cancer drugs Imatinib and Dasatinib on human chronic myeloid leukemia (K562) cell line. In this study the effect of Inecalcitol, Imatinib, and Dasatinib each drug alone and the combination of Imatinib and Dasatinib drugs separately with Inecalcitol, were estimated in vitro using MTT assay against K562 cell line. The concentrations that were studied for Inecalcitol, Imatinib, and Dasatinib ranged from (10nM to 100µM), (1nM to10µM), (0.001nM to10nM) respectively. The combination of Inecalcitol with Imatinib and Dasatinib drugs was evaluated in different trials, those trials were conducted using the same final concentrations of Imatinib and Dasatinib and different GI50 concentrations of Inecalcitol (0.5×, 1×, 1.5× and 2×GI50). In this study Inecalcitol, Imatinib, and Dasatinib showed a potent antiproliferative activity with GI50 values of (5.6 µM, 0.327 µM, and 0.446 nM) respectively. The combination of Imatinib and Dasatinib drugs separately with different Inecalcitol concentrations against the K562 cell line was tested in this study, and showed a significant growth inhibitory activity effect (p < 0.0001****) for both drugs, with optimal GI50 values of 0.58 nM and 0.00051 nM, respectively. Therefore, the combinations are recommended to reduce the dosage and side effects of Imatinib and Dasatinib drugs. This study showed a potent inhibitory effect of Inecalcitol (Vitamin D3 analogue) on K562 cell line, also it has showed the potentiation effect of Inecalcitol (Vitamin D3 analogue) when combined with antileukemic drugs Imatinib and Dasatinib on K562 cell line, in cell culture. Keywords: Cancer, Chronic myeloid leukemia, Inecalcitol, Imatinib, Dasatinib, K562 cell line.