Serologic and molecular evidence of Anaplasma phagocytophilum has been demonstrated in white-tailed deer (WTD; Odocoileus virginianus), and deer are an important host for the tick vector Ixodes scapularis. In this study, we describe experimental infection of WTD with A. phagocytophilum. We inoculated four WTD with a human isolate of A. phagocytophilum propagated in tick cells. Two additional deer served as negative controls. All inoculated deer developed antibodies (titers, ≥64) to A. phagocytophilum, as determined by an indirect fluorescent antibody test, between 14 and 24 days postinfection [p.i.]), and two deer maintained reciprocal titers of ≥64 through the end of the 66-day study. Although morulae were not observed in granulocytes and A. phagocytophilum was not reisolated via tick cell culture of blood, 16S reverse transcriptase nested PCR (RT-nPCR) results indicated that A. phagocytophilum circulated in peripheral blood of three deer through at least 17 days p.i. and was present in two deer at 38 days p.i. Femoral bone marrow from one deer was RT-nPCR positive for A. phagocytophilum at 66 days p.i. There was no indication of clinical disease. These data confirm that WTD are susceptible to infection with a human isolate of A. phagocytophilum and verify that WTD produce detectable antibodies upon exposure to the organism. Because adults are the predominant life stage of I. scapularis found on deer and because adult I. scapularis ticks do not transmit A. phagocytophilum transovarially, it is unlikely that WTD are a significant source of A. phagocytophilum for immature ticks even though deer have a high probability of natural infection. However, the susceptibility and immunologic response of WTD to A. phagocytophilum render them suitable candidates as natural sentinels for this zoonotic tick-borne organism.