STAT3 responds to cytokines in the microenvironment of the cell to orchestrate effector responses such as proliferation, inflammation, metastasis, and developmental programming. STAT3 is persistently active in many cancers, including those associated with the human gammaherpesvirus pathogens, Kaposi’s sarcoma-associated herpesvirus and Epstein Barr virus. A critical aspect of infection by these viruses is life-long maintenance of the viral genome as a latent episome, commonly in B cells, with the potential for reactivation or cellular transformation. STAT3 is at the molecular crossroads of innate and adaptive immunity, and in B cells it responds to IL-6, IL-10, and IL-21 and promotes high-affinity antibody production. We investigated the role of STAT3 in viral latency establishment in B cells by the murine gammaherpesvirus model pathogen, MHV68, in a newly infected animal. We infected mice with a B cell specific deletion of STAT3 (STAT3fl/fl;CD19-Cre) by intranasal and intraperitoneal routes. Following intranasal infection, establishment of viral latency was substantially impaired as assayed by colonization of the spleen and persistent infection of germinal center immunoglobulin class-switched B cells. The more direct intraperitoneal route allowed infection of germinal center B cells, but still showed a striking defect in latency establishment in class-switched and plasma cells. Regardless of route, persistence of MHV68 was dramatically reduced in the absence of STAT3. The lack of STAT3 did not impact acute infection of lungs, or productive infection in primary fibroblasts in culture, or in vitro reactivation from the few B cell reservoirs in the spleen at 16 dpi. Taken together, we identify STAT3 as a critical host determinant of gammaherpesvirus latency establishment in the animal host. For the first time our findings recognize STAT3 as a mediator of chronic gammaherpesvirus infection in B cells of infected animals, and provide a framework for mechanistic studies of the intrinsic role for STAT3.