Embryo implantation into maternal endometrium is critical for initiation and establishment of pregnancy, requiring developmental synchrony between endometrium and blastocyst. However, factors regulating human endometrial-embryo crosstalk and facilitate implantation remain largely unknown. Extracellular vesicles (EVs) are emerging as important mediators of this process. Here, we used a trophectoderm spheroid-based in vitro model mimicking the pre-implantation human embryo to recapitulate important functional aspects of blastocyst implantation. Functionally, human endometrial EVs, derived from hormonally-treated cells synchronous with implantation, were readily internalised by trophectoderm cells, regulating adhesive and invasive capacity of human trophectoderm spheroids. To gain molecular insights into mechanisms underpinning endometrial-EV mediated enhancement of implantation, quantitative proteomics revealed critical alterations in trophectoderm cellular adhesion networks (cell adhesion molecule binding, cell-cell adhesion mediator activity, and cell adherens junctions), and metabolic and gene expression networks) and the soluble-secretome from human trophectodermal spheroids. Importantly, we demonstrate transfer of endometrial EV-cargo proteins to trophectoderm to mediate changes in trophectoderm function. This is highlighted by correlation between endometrial-EVs, the trophectodermal proteome following EV uptake, and EV-mediated trophectodermal cellular proteome, important for implantation. This work provides an understanding into molecular mechanisms of endometrial EV-mediated regulation of human trophectoderm functions - fundamental in understanding human endometrium-embryo signalling during implantation. This article is protected by copyright. All rights reserved.