Introduction: Studies have reported that villous cytotrophoblasts (CTBs) undergo a partial epithelial to mesenchymal transition (EMT) when differentiating into extravillous cytotrophoblasts (EVTs). Epithelial splicing-regulatory protein 1 (ESRP1), an alternative splicing regulator, has been demonstrated to play important roles in the EMT process. Nevertheless, the roles of ESRP1 in the placentation remain undefined. Methods: ESRP1 expression in placental tissues throughout pregnancy was determined by immunohistochemistry and western blotting. The effect of ESRP1 knockdown by using small-interfering RNAs (siRNAs) on the phenotype of trophoblast cell line (HTR-8/SVneo) and villous explants was evaluated. Results: ESRP1 was localized within the CTBs, trophoblast columns, and EVTs located in the decidua. ESRP1 expression was changed during pregnancy, with the highest expression level in term placentae. ESRP1 knockdown significantly increased the migration and invasion of HTR-8/SVneo cells, as well as the outgrowth of EVTs from villous explants, without affecting cell proliferation. This enhanced effect was associated with the increased expression of N-cadherin, vimentin and CD44. Discussion: Our results suggested an important role for ESRP1 in regulating trophoblast migration and invasion during placentation.