Epithelial-mesenchymal transition (EMT) bestows cancer cells with motile and invasive properties. But for ovarian tissues, EMT plays a physiological role in the postovulatory repair of ovary surface epithelial (OSE) cells. Accumulating data indicated that 1α,25(OH)2D3 decreased both the migration and invasion of various cancer cells by suppressing EMT. However, it remains unclear whether 1α,25(OH)2D3 inhibits the process of EMT during different stages of oncogenic transformation in mouse OSE (MOSE) cells. In present study, a spontaneous malignant transformation model of MOSE cells at three sequential stages (early, intermediate and late) was established in vitro first and then subjected to 1α,25(OH)2D3 treatment to investigate the effect of 1α,25(OH)2D3 on the oncogenic transformation of MOSE cells. We found that 1α,25(OH)2D3 significantly reduced the proliferation and invasion of late malignant transformed MOSE (M-L cells) cells by inhibiting EMT both in vitro and in vivo, but not in intermediate transformed (M-I) cells. Importantly, we found that the levels of CYP24A1 in M-I cells were dramatically higher than that in M-L cells following treatment with 1α,25(OH)2D3. Furthermore, we demonstrated that, in both M-I and M-L cells with CYP24A1 knockdown, 1α,25(OH)2D3 suppressed the proliferation and invasion, and reduced the expression of N-cadherin, Vimentin, β-catenin and Snail. In addition, knockdown of CYP24A1 suppressed EMT by increasing E-cadherin while decreasing N-cadherin, Vimentin, β-catenin and Snail. These findings provide support for inhibiting CYP24A1 as a potential approach to activate the vitamin D pathway in the prevention and therapy of ovarian cancer.