The aim of the study was to evaluate the effects of single and double inseminations on the inflammation of the uterus in mares. Nine mix breed mares with endometrial biopsy scores grade I and IIA, aged 5-15, were used in the study. Two experiments were performed over two estrous cycles: (1) single insemination, (2) double insemination. All mares were used in both insemination group. In the single insemination cycles one insemination was performed 24 h after hCG injection, while in the double insemination cycles two inseminations were carried out 24 h and 48 h after the hCG injection. Endometrial biopsies were collected in both cycles immediately before (= 0), and 6, 30 and 54 h after the first insemination. Gene expressions of the inflammatory markers IL-1 beta, IL-6, IL-8, iNOS, SAA, COX-2 and CASP-3 and histopathological alterations of the endometrium were determined. The 6 h expression levels of IL-6 were higher when compared with 30 h and 54 h (P< 0.05). CASP-3 expression levels in single insemination cycles were higher at 0 h and 6 h when compared with 54 h (P< 0.05). In conclusion expression levels of the IL-1 beta,-6,-8, iNOS, SAA and COX-2 were not different between single or double insemination cycles. This study provides preliminary evidence to further characterize the changes in the expression of relevant genes in response to fresh semen.