Escherichia coli infection of the female reproductive tract is a significant cause of disease in humans and animals, but simple animal models are lacking. Here we report that vaginal inoculation of uropathogenic E. coli strains UTI89 and CFT073 in non-pregnant, estrogen-treated mice resulted in robust colonization of the vagina and uterine horns, whereas titers of the lab strain MG1655 were significantly lower. Non-estrogenized mice also became colonized, but there was more variation in titers. A dose of 104 colony-forming units (CFU) UTI89 was sufficient to result in colonization in all estrogenized mice, and we also observed bacterial transfer between inoculated and uninoculated estrogenized cage mates. UTI89 infection led to inflammation and leukocyte infiltration into the uterine horns as evidenced by tissue histology. Flow cytometry experiments revealed that neutrophil, monocyte and eosinophil populations were significantly increased in infected uterine horns. This model is a simple way to study host-pathogen interactions in E. coli vaginal colonization and uterine infection. There are immediate implications for investigators studying urinary tract infection using mouse models, as few E. coli are required to achieve reproductive colonization, resulting in an additional, underappreciated mucosal reservoir.