Updated project metadata. Extracellular vesicles (EVs) have emerged as key messengers in intercellular communications. However, the precise mechanisms of how recipient cells read EV messages are not clear. In this study, we investigated the roles of EV protein cargo, EV parent cell type, and recipient cell type in EV function using a human embryo implantation model. The JAr cell line served as an analog of human trophoblast cells, and EVs isolated from these cells were introduced into receptive endometrial epithelial cell analog RL95-2 cells to simulate EV-mediated embryo-maternal communication. Downregulation of Zinc-finger protein 81 (ZNF81) gene expression in RL95-2 cells in response to trophoblast EVs were used as a readout of JAr EV functionality. EVs from the non-trophoblastic cell line (HEK-293 cells) were used as a control to assess the specificity of trophoblast EV functionality in endometrial epithelial cells. Our results demonstrated that ZNF81 gene downregulation is a specific response of RL95-2 cells to JAr EVs, as HEK-293 cell EVs did not induce any gene expression changes. Furthermore, we conducted an analysis of the proteomic cargo profiles of JAr and HEK-293 cell EVs. Interestingly, JAr EVs exhibited a unique proteomic cargo profile compared to that of HEK EVs. Some of the proteins uniquely identified in JAr EVs are also known to play roles in embryo implantation. Moreover, JAr EVs were specifically enriched with important transcription factors that regulate ZNF81 gene expression. These findings contribute to our understanding of the specificity of EV functionality and cellular response in the context of embryo implantation.