Escherichia coli is a zoonotic bacterium that is important to both public health and livestock economics. To date, most studies of zoonotic E. coli transmission have been conducted in developed nations with industrialized agricultural economies. In this study, E. coli bacteria were collected from people and livestock in two communities in rural western Uganda in order to investigate patterns of interspecific bacterial transmission in a developing rural economy characterized by very close human-livestock associations. Six hundred seventy-two E. coli isolates were genotyped using repetitive element-PCR (Rep-PCR) fingerprinting, and genetic distances between populations of bacteria from different hosts and locations were calculated. Genetic distances between human and livestock bacteria were generally very low, indicating high rates of bacterial gene flow among host species. Bacteria from humans and livestock in the same communities were virtually indistinguishable genetically. Data from surveys administered at the time of sample collection showed that people who did not regularly wash their hands before eating harbored bacteria approximately twice as similar genetically to bacteria of their livestock as did people who regularly washed their hands before eating. These results suggest that both rates of human-livestock interactions and patterns of human hygiene affect human-livestock bacterial transmission in this setting. This conclusion has implications not only for human and livestock health in subsistence-based agricultural economies but also for the emergence of zoonotic diseases out of such areas as a result of increasing globalization.