Placental vascular dysfunction has been linked to insufficiency/deficiency of maternal vitamin D levels during pregnancy. In contrast, sufficient maternal vitamin D levels have shown beneficial effects on pregnancy outcomes. To study the role of vitamin D in pregnancy, we tested our hypothesis that vitamin D exerts beneficial effects on placental vasculature. We examined expression of CYP2R1, CYP27B1, vitamin D receptor (VDR), and CYP24A1 in placental vascular smooth muscle cells (VSMCs) in response to 1,25(OH)₂D₃. We found that VDR expression was inducible, CYP27B1 expression was dose-dependently down-regulated, and CYP24A1 expression was dose-dependently up-regulated in cells treated with 1,25(OH)₂D₃. These data suggest a feedback autoregulatory system of vitamin D existing in placental VSMCs. Using a VSMC/collagen-gel contraction assay, we evaluated the effect of 1,25(OH)₂D₃ on placental VSMC contractility. We found that, similar to losartan, 1,25(OH)₂D₃ could diminish angiotensin II-induced cell contractility. The mechanism of 1,25(OH)₂D₃-mediated VSMC relaxation was further explored by examination of Rho-associated protein kinase 1 (ROCK1)/phosphorylation of myosin phosphatase target subunit 1 (MYPT1) pathway molecules. Our results showed that p-MYPT1^Thr853 and p-MYPT1^Thr696 were undetectable. However, p-MYPT1^Ser507, but not p-MYPT1^Ser668, was significantly up-regulated in cells treated with losartan plus angiotensin II. Similar effects were also seen in cells treated with 1,25(OH)₂D₃ plus angiotensin II or 1,25(OH)₂D₃ plus losartan plus angiotensin II. Because MYPT1 serine phosphorylation could activate myosin light chain phosphatase (MLCP), and MLCP activation is an important regulatory machinery of smooth muscle cell relaxation, up-regulation of MYPT1^Ser507 phosphorylation could be a mechanism of vitamin D and/or losartan mediated placental VSMC relaxation.