Bacterial infection of the uterus occurs after parturition in approximately 40% of dairy cows and reduces fertility after the resolution of disease. To understand the mechanisms of uterine infection associated infertility, we induced uterine disease in virgin Holstein heifers. Heifers received either bacterial infusion (n = 4) or control vehicle infusion (n = 6) and were monitored for 95 d. All heifers were free from disease before experimentation. Estrous cycles were synchronized and uterine inoculation was performed on d 2 of the estrous cycle. Endometrial scarification was implemented before inoculation of vehicle or the bacteria, which were isolated from clinical cases of postpartum uterine disease in dairy cows (Escherichia coli MS499 + Trueperella pyogenes MS249). Animal health and disease development was evaluated by rectal temperature, vaginal discharge, visualization of uterine content by transrectal ultrasonography, plasma concentrations of inflammatory mediators, and hematology until reproductive tracts were collected at d 95. Data were analyzed by mixed models using the MIXED procedure of SAS. Bacterial infusion increased vaginal discharge above grade 2 at d 4, whereas controls remained below grade 1. Vaginal discharge scores remained greater in bacterial infusion heifers compared with controls until d 10. Ultrasound revealed the presence of echogenic pus in the uterus following bacterial infusion. Bacterial infusion increased (P = 0.02) circulating lymphocytes on d 14 and reduced (P = 0.05) red blood cells on d 18 compared with control. Rectal temperature, general health, and appetite did not differ between treatments. No differences were observed in plasma IL-6, TNFα or haptoglobin concentrations between treatments. As indicated by increased vaginal discharge after bacterial infusion, this model successfully induces clinical endometritis in virgin heifers. This model isolates the negative effects of uterine infection on fertility from confounding factors that can occur during the postpartum period in dairy cows. Supported by the Eunice Kennedy Shriver NICHD R01HD084316.