Immune dysregulation reduces endometrial receptivity and contributes to infertility. Mast cells are key immune cells in the endometrium, and their activation may disrupt inflammation. Vitamin D, as an immunomodulator, may act by modulating mast cell activity. In this experimental study, 20 adult female NMRI rats were randomly assigned to control and vitamin D-treated groups. The experimental group received 1000 IU of vitamin D per kilogram body weight daily for three weeks. In both groups, pseudopregnancy was induced, and 3.5 days later, before implantation, tissue was sampled from the middle third of the uterine horn and histological sections were prepared. Toluidine blue staining and histopathological examination were performed. Mast cell number and degranulation status were evaluated in three patterns: granulated, partially granulated, and degranulated. Data were analyzed using generalized estimating equations with a Poisson distribution. Vitamin D reduced the mean number of endometrial mast cells, but the reduction was not significant (IRR=0.87, CI=0.58-1.31, p=0.508). The incidence rate of granulated mast cells was 0.84 and was not significant (IRR=0.84, 95% CI=0.54-1.32, p=0.455). Partially granulated mast cells increased, but this change was also not significant (p=0.677). The incidence rate of degranulated mast cells in the vitamin D group was 0.58, indicating a decrease, but it was not significant (IRR=0.58, 95% CI=0.17-1.97, p=0.386). Overall, vitamin D did not produce a statistically significant difference; however, it may modulate endometrial inflammation in this model by reducing mast cell activation and help create a favorable implantation environment. Confirmation of this hypothesis requires further studies and functional markers.