Despite the use of adjuvant therapies, the cumulative proportion of live births remains at ~40%. Accumulating data show that low pregnancy rates, even in the presence of high fertility rates, are due to implantation failure. The present study aimed to identify and construct a profile of proteins that react with preimplantation factor (PIF) and to provide an understanding into the molecular mechanisms by which PIF promotes trophoblast invasion. Cytoplasmic proteins were immunoprecipitated with biotin?labeled synthetic PIF or intralipid and scrambled PIF (PIFscr). The protein profiles were analyzed using isobaric tags for relative and absolute quantification coupled with mass spectrometry. Immunoprecipitation and western blot analyses were used to assess the interactions between PIF and myosin heavy chain 10 (MYH10) and heat shock protein family D1. Small interfering RNA?based silencing was performed to examine the function of MYH10. In the results of the present study, 21 proteins were identified with interactions with PIF. The immunoprecipitation and western blot analyses revealed an interaction between PIF and MYH10. Silencing of the expression of MYH10 in HEC?1?B cells significantly attenuated cell migration and invasion capacities. These data support the conclusion that MYH10?mediated cell migration and invasion act in conjunction with PIF to promote the trophoblast invasion procedure.