Wildlife are an important component in the vector-host-pathogen triangle of livestock diseases, as they maintain biological vectors that transmit pathogens and can serve as reservoirs for such infectious pathogens. Babesia bovis is a tick-borne pathogen, vectored by cattle fever ticks, Rhipicephalus spp., that can cause up to 90% mortality in naive adult cattle. While cattle are the primary host for cattle fever ticks, wild and exotic ungulates, including white-tailed deer (WTD), are known to be viable alternative hosts. The presence of cattle fever tick populations resistant to acaricides raises concerns regarding the possibility of these alternative hosts introducing tick-borne babesial parasites into areas free of infection. Understanding the B. bovis reservoir competence of these alternative hosts is critical to mitigating the risk of introduction. In this study, we tested the hypothesis that WTD are susceptible to infection with a B. bovis strain lethal to cattle. Two groups of deer were inoculated intravenously with either B. bovis blood stabilate or a larval extract supernatant containing sporozoites from infected R. microplus larvae. The collective data demonstrated that WTD are neither a transient host nor reservoir of B. bovis. This conclusion is supported by the failure of B. bovis to establish an infection in deer regardless of inoculum. Although specific antibody was detected for a short period in the WTD, the PCR results were consistently negative at multiple time points throughout the experiment and blood from WTD that had been exposed to parasite, transferred into naïve recipient susceptible calves, failed to establish infection. In contrast, naïve steers inoculated intravenously with either B. bovis blood stabilate or the larval extract supernatant containing sporozoites rapidly succumbed to disease. These findings provide evidence that WTD are not an epidemiological component in the maintenance of B. bovis infectivity to livestock.