The white-footed mouse, Peromyscus leucopus (Rafinesque), is a reservoir for the Lyme disease spirochete Borrelia burgdorferi sensu stricto in the eastern half of the United States, where the blacklegged tick, Ixodes scapularis Say (Acari: Ixodidae), is the primary vector. In the Midwest, an additional Lyme disease spirochete, Borrelia mayonii, was recorded from naturally infected I. scapularis and P. leucopus. However, an experimental demonstration of reservoir competence was lacking for a natural tick host. We therefore experimentally infected P. leucopus with B. mayonii via I. scapularis nymphal bites and then fed uninfected larvae on the mice to demonstrate spirochete acquisition and passage to resulting nymphs. Of 23 mice fed on by B. mayonii-infected nymphs, 21 (91%) developed active infections. The infection prevalence for nymphs fed as larvae on these infected mice 4 wk post-infection ranged from 56 to 98%, and the overall infection prevalence for 842 nymphs across all 21 P. leucopus was 75% (95% confidence interval, 72–77%). To assess duration of infectivity, 10 of the P. leucopus were reinfested with uninfected larval ticks 12 wk after the mice were infected. The overall infection prevalence for 480 nymphs across all 10 P. leucopus at the 12-wk time point was 26% (95% confidence interval, 23–31%), when compared with 76% (95% confidence interval, 71–79%) for 474 nymphs from the same subset of 10 mice at the 4-wk time point. We conclude that P. leucopus is susceptible to infection with B. mayonii via bite by I. scapularis nymphs and an efficient reservoir for this Lyme disease spirochete.