Tyrosine phosphorylation plays a major role in controlling many biological processes in different cell types. Src family kinases (SFK) are one of the most studied tyrosine kinases and can mediate a variety of signaling pathways. However, little is known about the expression of SFK in human term placenta and their implication in trophoblasts differentiation. Therefore, we examined the expression profile of SFK members along the days of culture and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultured in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that resemble phenotypically to mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). In this study, we showed that trophoblasts expressed all SFK members and some of them are expressed as different splice variants. Moreover, using real-time PCR, this study showed two different expression profiles of SFK in human trophoblasts along the days of culture. In addition, the protein level and phosphorylation status of Src were evaluated using specific antibodies. Src was rapidly phosphorylated at tyrosine 416 (Y416) and dephosphorylated at Y527 after FBS addition. Surprisingly, inhibition of SFK by PP2 or herbimycin A had different effects on trophoblasts differentiation. While herbimycin A inhibited morphological and hormonal differentiation, PP2 stimulated hormonal differentiation and inhibited cell adhesion and spreading with no effect on cell fusion. In summary, this study showed that SFK play different roles in trophoblasts differentiation, probably depending on SFK members activated. Thus, this study improves our knowledge in the understanding of pathology related to impaired trophoblasts differentiation such as preeclampsia and trophoblasts neoplasm.