Cytomegalovirus (CMV) infects a majority of the human population and establishes a life-long persistence. CMV infection is usually asymptomatic but the virus carries pathogenic potential and causes severe disease in immunocompromised individuals. T-cell-mediated immunity plays an essential role in control of CMV infection and adoptive transfer of CMV-specific CD8+ T cells restores viral immunity in immunosuppressed patients but a role for CD4+ T cells remains elusive. Here, we analyzed in adoptive transfer studies the features and antiviral functions of virus-specific CD4+ T cells during primary murine CMV (MCMV) infection. MCMV-specific CD4+ T cells expanded upon MCMV infection and displayed an effector phenotype and function. Adoptive transfer of in vivo activated MCMV-specific CD4+ T cells to immune-compromised mice was protective during pathogenic MCMV infection and IFN-γ was a crucial mediator of this protective capacity. Moreover, co-transfer of low doses of both MCMV-specific CD4+ T cells and CD8+ T cells synergized in control of lytic viral replication in immune-compromised mice. Our data reveal a pivotal antiviral role for virus-specific CD4+ T cells in protection from pathogenic CMV infection and provide evidence for their antiviral therapeutic potential. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.