Decidual NK (dNK) cells, identified as CD56brightCD16-CD3-, account for ˜70% of lymphocytes within the uterine wall during early pregnancy. Accumulating evidence suggests that tight interactions between placental trophoblasts and dNK cells are critical for trophoblast cell differentiation. However, the underlying mechanism remains to be explored in detail. In the present study, conditioned medium (CM) was collected from cultured primary human dNK cells. Primary cytotrophoblasts (CTBs) or the human trophoblast cell line HTR8/SVneo was treated with dNK-CM and co-cultured with human umbilical vein endothelial cells (HUVECs) in a three-dimensional Matrigel scaffold, and the formation of tube structures was dynamically monitored with live cell imaging. Trophoblast invasion was analyzed with a transwell invasion assay. The data demonstrated that the treatment of HTR8/SVneo cells or CTBs with dNK-CM remarkably promoted trophoblast invasion and tube formation in the presence of HUVECs. The epithelial marker E-cadherin was reduced, while the expression of endothelial markers NCAM, VE-cadherin and integrin β1 was significantly promoted in the HTR8/SVneo cells upon treatment with dNK-CM. Antibody blocking experiments revealed that the dNK cells promoted trophoblast invasion through the production of IL-8 and HGF, and they induced trophoblast differentiation toward endothelial phenotype by producing VEGF-C and HGF. These results provide new evidence to clarify the finely tuned interactions between trophoblasts and dNK cells at the maternal-fetal interface.