Establishment of a successful pregnancy requires synergistic communication between the endometrium and the blastocyst during the pre-implantation phase. Endometrial exosomes released into the uterine microenvironment are proposed to play essential role in the implantation process. In this study, we investigated the proteomic profiles of endometrial exosomes across the menstrual cycle and examined the effects of exosomes on trophoblast function. Mass spectrometry was used to study the highly purified exosomes isolated from ECC1 endometrial epithelial cells, treated with estrogen and progesterone. From a total of 1073 exosomal proteins identified, 684 were found common, while 258 and 131 proteins were uniquely enriched in response to estrogen and progesterone respectively. Functionally, 24- hour live cell imaging showed a progressive accumulation of exosomes in HTR8 trophoblast cells, resulting an increase in adhesion response of 24 % (p < 0.001). Western blot analyses of endometrial exosomes and HTR8 cell co-culture suggested that focal adhesion kinase signaling pathway may be involved in adhesion response of trophoblast cells. This study is the first to demonstrate that cargo proteins packaged within endometrial exosomes are important during pre-implantation and may offer new avenues to improve receptivity and pregnancy outcomes.