Human adenovirus serotype 7 (HAdV-7), belonging to species B, has caused severe lower respiratory tract diseases and even deaths recently. However, no adenovirus vaccine or therapeutic is available thus far. In this study, a HAdV-7-specific human monoclonal antibody (HMAb), 3-3E, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of HAdV-7-infected patients showed potent HAdV-7 neutralization activity. The results showed HMAb 3-3E only binds to the hexon protein of intact HAdV-7 or the recombinant hexon protein and it does not bind to other intact virion particles. This could mean the antibody recognizes a conformational epitope of the hexon protein. Further, HMAb 3-3E potently neutralized HAdV-7 in vitro at low concentrations. In vivo studies showed HMAb 3-3E protected from HAdV-7 infection in a murine model. Therefore, HMAb 3-3E is promising as a safe and effective prophylactic and therapeutic treatment for HAdV-7 infection.