Infections with herpesviruses, including human roseoloviruses, have been proposed to cause autoimmune disease, but defining a causal relationship and mechanism has been difficult due to the ubiquitous nature of infection and development of autoimmunity long after acute infection. Murine roseolovirus (MRV) is highly related to human roseoloviruses. Herein we show that neonatal MRV infection induced autoimmune gastritis (AIG) in adult mice in the absence of ongoing infection. MRV-induced AIG was dependent on replication during the neonatal period and was CD4+ T cell and IL-17 dependent. Moreover, neonatal MRV infection was associated with development of a wide array of autoantibodies in adult mice. Finally, neonatal MRV infection reduced medullary thymic epithelial cell numbers, thymic dendritic cell numbers, and thymic expression of AIRE and tissue-restricted antigens, in addition to increasing thymocyte apoptosis at the stage of negative selection. These findings strongly suggest that infection with a roseolovirus early in life results in disruption of central tolerance and development of autoimmune disease.