Rabies has rarely been described in Xenarthra, and rabies vaccine response has not been documented. A southern tamandua (Tamandua tetradactyla) presented with nonspecific clinical signs and was euthanatized. Subsequently, immunohistochemistry and RT-PCR confirmed a rabies diagnosis. Following these tests, a group of eight captive tamanduas were vaccinated with a killed rabies vaccine, and titers were measured at the time of vaccination and 23 d later. One animal had day 0 titers suggestive of previous vaccination or exposure. All animals had detectable neutralizing rabies virus antibody titers after vaccination, but one animal failed to meet the World Organization for Animal Health's definition for adequate vaccination (≥0.5 IU/ml), and two other animals had low antibody titers (0.56 and 0.6 IU/ml). Rabies should be considered as a possible cause of illness in tamanduas, and rabies vaccination may be a useful preventative measure when anthropic interaction through medical care or ambassador roles is occurring.