Trophoblasts are important parts of the placenta and exert vital roles in the maternal-foetal crosstalk, and sufficient trophoblasts migration and invasion is critical for embryo implantation and normal pregnancy. Macrophages, as the major components of decidual microenvironment at maternal-foetal interface, can interact with trophoblasts to participate in the regulation of normal pregnancy. Previously, our group have demonstrated that trophoblasts could induce macrophages polarization to M2 subtype by secreting interleukin-6 (IL-6); however, the understanding of macrophages regulating the migration and invasion of trophoblasts is limited. In the present study, we used the co-cultured model to further investigate the effects of macrophages on trophoblasts migration and invasion. Our results showed that co-culture with macrophages promoted epithelial-to-mesenchymal transition (EMT) of trophoblasts, thereby enhancing their migrative and invasive abilities. Further experiments revealed that M2 macrophage-derived G-CSF was a key factor, which promoted the EMT, migration and invasion of trophoblasts via activating PI3K/Akt/Erk1/2 signalling pathway. Clinically, G-CSF was highly expressed in placental villous tissues of normal pregnancy patients compared to patients with recurrent spontaneous abortion, and its expression level was significantly correlation with EMT markers. Taken together, these findings indicate the important role of M2 macrophages in regulating trophoblasts EMT, migration and invasion, contributing to a new insight in concerning the crosstalk between macrophages and trophoblasts in the establishment and maintenance of normal pregnancy.