The human endometrium undergoes substantial remodelling in each menstrual cycle to become receptive to an implanting embryo. Abnormal endometrial receptivity is one of the major causes of embryo implantation failure and infertility. MicroRNAs are dysregulated in the endometrial epithelium of women with infertility at the receptive phase. However, there is limited evidence on how individual dysregulated microRNAs lead to endometrial dysfunction. In this study, we aimed to determine the function of microRNA-124-3p on the adhesive capacity of endometrial epithelial cells, which is essential for securing firm embryo attachment to the endometrial surface. qPCR and in situ hybridization were used to determine the expression and localisation of microRNA-124-3p in fertile and infertile human endometrium. To test the regulation of microRNA-124-3p on cell adhesion, primary human endometrial epithelial cells and Ishikawa cells (a receptive endometrial epithelial cell line) were subjected to trophoblast spheroid (blastocyst surrogate) adhesion assay and xCELLigence, respectively. We optimized a trypsin “shaving” condition to cleave Ishikawa cell surface-accessible proteins following control or microRNA-124-3p overexpression. Mass spectrometry was applied to determine profile of cleaved proteins. Our results showed that MicroRNA-124-3p was abnormally increased in the endometrial luminal epithelium of women with unexplained infertility during the receptive phase, compared to fertile controls. Functionally, microRNA-124-3p overexpression in primary human endometrial epithelial cells and Ishikawa cells significantly impaired their adhesive capacity compared to respective controls. Incubation of trophoblast spheroids with trypsin shaving media significantly impacted their adhesion to untreated Ishikawa monolayers. Mass spectrometry analysis of the shaved surface proteins from Ishikawa cells (both control and microRNA-124-3p overexpressed) identified a total of 298 proteins, with 17 of them being significantly reduced after microRNA-124-3p overexpression. Overall, our findings suggest that microRNA-124-3p plays a role in blocking endometrial receptivity by reducing epithelial cell adhesion.