CYP27B1 hydroxylates 25-hydroxyvitamin D3 in position C1α into biologically active 1,25-dihydroxyvitamin D3, calcitriol. CYP27B1 is expressed in normal tissues and tumors. Since calcitriol indicates anticancer activities and CYP27B1 expression can be deregulated during malignant progression, we analyzed its expression in ovarian cancers in relation to pathomorphological features of tumors and overall survival (OS). Expression of CYP27B1 was evaluated in 61 ovarian tumors, 18 metastases and 10 normal ovaries. Normal ovarian epithelium showed the highest levels CYP27B1 with a significant decrease in its expression in ovarian cancers. Both poorly differentiated primary tumors and metastases showed the lowest level of CYP27B1 expression, while non-metastasizing tumors showed a higher CYP27B1 level than tumors that developed metastases. The expression of CYP27B1 was positively correlated with a lower proliferation rate, lower dynamism of tumor growth and tumor infiltrating lymphocyte response. Furthermore, CYP27B1 expression was negatively correlated with tumor cell modeling of their microenvironment. CYP27B1 expression was also associated with longer OS time. In summary, our results suggest that local expression of CYP27B1 in ovarian tumor cells can modify their behavior and promote a less aggressive phenotype by affecting local concentrations of active of vitamin D levels within the tumor microenvironment.