Lyme borreliosis is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato group, which are transmitted by Ixodes tick species living in the temperate climate zones of the Northern Hemisphere. The clinical manifestations of Lyme borreliosis are diverse and treated with oral or intravenous antibiotics. Lyme borreliosis is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato group, which are transmitted by Ixodes tick species living in the temperate climate zones of the Northern Hemisphere. The clinical manifestations of Lyme borreliosis are diverse and treated with oral or intravenous antibiotics. In some patients, long-lasting and debilitating symptoms can persist after the recommended antibiotic treatment. The etiology of such persisting symptoms is under debate, and one hypothesis entails persistent infection by a subset of spirochetes after antibiotic therapy. Here, we review and appraise the experimental evidence from in vivo animal studies on the persistence of B. burgdorferi sensu lato infection after antibiotic treatment, focusing on the antimicrobial agents doxycycline and ceftriaxone. Our review indicates that some in vivo animal studies found sporadic positive cultures after antibiotic treatment. However, this culture positivity often seemed to be related to inadequate antibiotic treatment, and the few positive cultures in some studies could not be reproduced in other studies. Overall, current results from animal studies provide insufficient evidence for the persistence of viable and infectious spirochetes after adequate antibiotic treatment. Borrelial nucleic acids, on the contrary, were frequently detected in these animal studies and may thus persist after antibiotic treatment. We put forward that research into the pathogenesis of persisting complaints after antibiotic treatment for Lyme borreliosis in humans should be a top priority, but future studies should most definitely also focus on explanations other than persistent B. burgdorferi sensu lato infection after antibiotic treatment.