During the estrous cycle, the endometrium epithelium experiences marked cellular structural changes. For fertilization to proceed, maintenance of an adequate uterine environment by ovarian hormones is essential. Epithelial cells lining the uterine lumen are associated with each other by tight junctions (TJs), which regulate the passage of ions and molecules through the paracellular pathway. The aim of the present study was to assess by confocal immunofluorescence the distribution pattern of the TJ proteins ZO-1, occludin, and claudins 1–7 in the rat uterus during the estrous cycle. Our results reveal that on proestrus, the day when mating takes place, ZO-1, occludin, and claudins 1 and 5 are located in the TJs, while claudins 3 and 7 display a basolateral distribution. In contrast, on metestrus day, when no sexual mating occurs and the uterine lumen is devoid of secretions, none of these proteins were detected in the TJ region, and only a diffuse cytosolic staining was observed for some of the proteins. On estrus and diestrus days, an intermediate situation was encountered, since ZO-1 localized in the TJs, whereas occludin was no longer detectable in the TJs. The distribution of claudins during these stages varied from the lowermost portion of the basolateral membrane to its apex. In conclusion, the results show that the protein composition of TJs present in the luminal epithelial cells of the uterus changes during the different days of the estrous cycle, and suggest that the expression of TJ proteins participates in providing an adequate environment for a successful fertilization.