Objective To analyze the antiviral effect of molnupiravir against influenza virus in vitro and in vivo. Methods The antiviral ability of molnupiravir against influenza virus strain H1N1 PR8 was detected in vitro and in vivo. Human non-small cell lung cancer cell line (A549 cells) was used as an in vitro model of PR8 infection. The antiviral effects of molnupiravir on virus replication, protein synthesis, and viral particle formation were analyzed using real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), Western blot (WB) assay, and plaque assay. PR8 nose-dripping infected C57BL/6 female mice were used as an in vivo infection model. The antiviral ability of molnupiravir was evaluated by detecting viral load, pathological changes, and immunohistochemistry in the control group, administration group, inoculation group, and inoculation-administration group. Results In vitro test results showed that molnupiravir had no significant inhibitory effect on influenza virus replication, protein synthesis, and virus particle formation (all P>0.05). In vivo test results showed that compared with mice infected with H1N1 alone, the viral load in the lung tissue of mice treated with molnupiravir declined significantly, and the extent of pathological changes was milder. Immunohistochemical detection showed a significant weakening in nuclear protein (NP) antigen signal, and the expression levels of interferon (IFN)-α, interleukin (IL)-4, and IL-6 in the lungs were lower (all P<0.01). Conclusion As a precursor with antiviral activity, molnupiravir can not exert antiviral activity in lung-derived cells cultured in vitro, but can be transformed into an active form in the host, which significantly decreases the ability of H1N1 influenza virus to proliferate in the lungs and thereby alleviates the da-mage of virus to lung tissue.