The human endometrium undergoes cycle dependent changes and is only receptive to an implanting blastocyst within a narrow window of 2-4 days in the mid-secretory phase. Recent single cell sequencing of human endometrium across the menstrual cycle has identified an abrupt and discontinuous transcriptomic activation in the epithelia in the mid-secretory phase. Such transcriptomic and accordingly functional changes require delicate interplay between a diversity of factors including cytokines and signaling pathways. The Notch signaling pathway members are expressed in human endometrium. We have previously demonstrated that Notch ligand Jagged1 (JAG1) localizes in the endometrial luminal epithelium (LE) and is abnormally reduced in infertile women during receptivity. However, the functional consequences of reduced JAG1 production on endometrial receptivity to implantation of the blastocyst are unknown. This study aimed to determine the role of JAG1 in regulating endometrial receptivity in humans and mice. Knockdown of JAG1 in both primary human endometrial epithelial cells and Ishikawa cells (endometrial epithelial cell-line) significantly reduced their adhesive capacity to HTR8/SVneo (trophoblast cell-line) spheroids. We confirmed that in human endometrial epithelial cells, JAG1 interacted with Notch Receptor 3 (NOTCH3) and knockdown of JAG1 significantly reduced the expression of Notch signaling downstream target HEY1 and classical receptivity markers. Knockdown of Jag1 in mouse LE significantly impaired blastocyst implantation. Via a customized RT2 Profiler PCR array, we identified ten genes (related to tight junction, infertility and cell adhesion) that were differentially expressed by Jag1 knockdown in LE in mice. Further analysis of the tight junction family members in both species revealed that JAG1 altered the expression of tight junction components only in mice. Together, our data demonstrated that JAG1 altered endometrial epithelial cell adhesive capacity and regulated endometrial receptivity in both humans and mice likely via different mechanisms.