Abstract: Objective To investigate the effect of vitamin D on microRNA-21 (miR-21) expression and migration and invasion of human placental trophoblast cells. Methods The changes in the expression of miR-21 were detected using RT-qPCR in HTR-8/ SVneo cells following stimulation by vitamin D at different doses for 24, 48 and 72 h. HTR-8/SVneo cells transfected with miR-21 mimic or inhibitor with or without vitamin D treatment were examined for changes in cell migration and invasion abilities using Transwell assay, and Western blotting was used to detect protein expressions of E-cadherin, fibronectin, and MMP9. Results Vitamin D obviously inhibited the expression of micoRNA-21 in HTR-8/SVneo cells in a concentration-and time- dependent manner. Transfection with the miR-21 mimic significantly inhibited the migration and invasion of HTR-8/ SVneo cells, and this inhibitory effect was abolished by treatment with vitamin D; transfection with miR-21 inhibitor obviously promoted the migration and invasion of HTR-8/SVneo cells, and these effects were not significantly affected by vitamin D treatment. Conclusion Vitamin D may promote trophoblast cell migration and invasion to accelerate the development of preeclampsia by down-regulating the expression of miR-21.