Human adenovirus (HAdV) infection is a major cause of respiratory disease, yet no antiviral drugs have been approved for its treatment. Herein, we evaluated the antiviral and anti-inflammatory effects of cyclin-dependent protein kinase (CDK) inhibitor indirubin-3′-monoxime (IM) against HAdV infection in cells and a transgenic mouse model. After evaluating its cytotoxicity, cytopathic effect reduction, antiviral replication kinetics, and viral yield reduction assays were performed to assess the anti-HAdV activity of IM. Quantitative real-time polymerase chain reaction (qPCR), quantitative reverse transcription PCR (qRT-PCR), and western blotting were used to assess the effects of IM on HAdV DNA replication, transcription, and protein expression, respectively. IM significantly inhibited HAdV DNA replication as well as E1A and Hexon transcription, in addition to significantly suppressing the phosphorylation of the RNA polymerase II C-terminal domain (CTD). IM mitigated body weight loss, reduced viral burden, and lung injury, decreasing cytokine and chemokine secretion to a greater extent than cidofovir. Altogether, IM inhibits HAdV replication by downregulating CTD phosphorylation to suppress viral infection and corresponding innate immune reactions as a promising therapeutic agent.