OBJECTIVE: Over the last few years, vitamin D deficiency (VDD) was inconsistently reported to be linked to the development of preeclampsia. Osteopontin is an adhesion molecule necessary for trophoblastic invasion, a process which is defective in preeclampsia. As vitamin D was shown to affect osteopontin expression in different tissues, this study investigated this relation in preeclampsia. PATIENTS AND METHODS: Gestational age-matching (32–40 weeks) controls (n=51) and preeclamptic pregnancies (n=51) were enrolled in this case–control study. Preeclampsia was subdivided into mild, early-onset, and late-onset severe preeclampsia (EOSPE and LOSPE). Serum levels of vitamin D were assessed. The intensity and distribution of osteopontin were immunohistochemically evaluated in extravillous cytotrophoblasts. RESULTS: Vitamin D deficiency was widely prevalent among the women studied (90.2%, n=92). Both VDD and osteopontin accumulation were more prominent in preeclampsia versus controls (P<0.001 for both), in EOSPE and LOSPE versus controls before and beyond 34 weeks, respectively (P<0.001) and in severe vs. mild preeclampsia (P=0.004 and 0.03 for VDD and osteopontin). Vitamin D levels correlated significantly with osteopontin expression, both in preeclampsia (r=−0.592043, P<0.001) and controls (r=−0.339094, P=0.008). Multiple regression analysis confirmed an association between osteopontin expression and VDD, independent of the gestational age, in EOSPE and LOSPE (P=0.03 vs. 0.77 and P=0.02 vs. 0.38, respectively). In mild preeclampsia and controls, both VDD and gestational age affected osteopontin expression. CONCLUSION: The correlation between VDD and higher expression of osteopontin in extravillous trophoblasts may represent one of the underlying mechanisms linking vitamin D to preeclampsia.