Human adenovirus (HAdV) infection is a frequent and potentially severe complication following allogeneic stem cell transplantation in children. Because treatment with antiviral drugs is often ineffective, adoptive transfer of donor-derived HAdV-specific T cells able to control viral replication of HAdV of multiple serotypes may be an option for therapy. In healthy donors, predominantly HAdV-specific T cells expressing CD4 are detected. In this study, a preclinical in vitro model was used to measure the antiviral effect of HAdV-specific CD4+ T cells. CD4+ HAdV-specific T cell clones restricted by HLA class II molecules were generated and most of these clones recognized conserved peptides derived from the hexon protein. These cross-reactive T cell clones were able to control viral replication of multiple serotypes of HAdV in EBV-transformed B cells (B-LCL), melanoma cells (MJS) and primary bronchial epithelial cells through cognate interaction. The HAdV-specific CD4+ T cell clones were able to specifically lyse infected target cells using a perforin-dependent mechanism. Antigenic peptides were also presented to the CD4+ T cell clones when derived from endogenously produced hexon protein. Together, these results show that cross-reactive HAdV-specific CD4+ T cells can control replication of HAdV in vitro and provide a rationale for the use of HAdV-specific T cells in adoptive immunotherapy protocols for control of life-threatening HAdV-infections in immunocompromised patients.