The Ca2+-selective transient receptor potential vanilloid 6 (TRPV6) channel plays a fundamental role in the mouse female and male reproductive system. In mouse endometrium the TRPV6 protein is highly expressed in epithelial cells (MEECs). Using Ca2+ imaging and patch clamp analysis we found that TRPV6 contributes to the cytosolic Ca2+ level, more specifically to the Ca2+ influx into the MEECs. As compared to wild-type cells, Trpv6-deficient MEECs reveal a significantly reduced frequency of spontaneous cytosolic Ca2+ oscillations, shown in both isolated cells and the complete uterus in situ. Noteworthy, in MEECs we here show for the first time distinct endogenous TRPV6-dependent whole-cell currents detected from primary cells. Our results disclose a previously unknown physiological role for TRPV6 in the regulation of endometrial Ca2+ homeostasis and its impact on female mouse fecundity, representing a basis for further investigation of reproductive impairments e.g. associated with defective Ca2+ regulation in women.