Low reproductive performance of buffaloes is a major problem faced by breeders. Anestrus and repeat breeding are the most common reproductive disorders in buffaloes. In this study, 25 buffalo-cows (normal cyclic, n = 9, pluriparous; anestrus, n = 7, nulliparous and repeat breeders, n = 9, pluriparous) were used to assess some biochemical and immunohistochemical changes in those that had reproductive disorders in comparison with normal cyclic animals. Serum calcium (Ca) and vitamin D (25(OH)D) levels were analyzed. Also, serum progesterone (P4) and estradiol (E2) levels were measured to assess the animals’ reproductive statuses. Additionally, immunohistochemical expression of calcium-binding protein-D9k (CaPB-9k) and vitamin D receptor (VDR) in the uterine tissues of all examined animals were evaluated. It was interesting that both biochemical and immunohistochemical results seem to correlate well. Both anestrus and repeat breeder buffalo-cows showed significant (p < 0.01) low serum levels of Ca and 25(OH)D as well as P4 and E2 in comparison with normal cyclic animals. Immunohistochemically, CaBP-9k and VDR immunoreactions were limited to the endometrial epithelia of all examined animals. But, both anestrus and repeat breeder buffalo-cows showed remarkable lower uterine expressions for CaBP-9k and VDR in comparison with normal cyclic ones. In conclusion, this study refers to the potential necessary role of Ca, 25(OH)D, CaBP-9k, and VDR in the continued fertility of buffalo-cows as well as, the endometrial viability. Therefore, our results suggest a link between the incidence of anestrous or repeat breeding in buffalo-cows and the low serum levels of Ca and 25(OH)D and/or low expression of uterine CaBP-9k and VDR. © 2020, Springer-Verlag London Ltd., part of Springer Nature.