Human metapneumovirus (hMPV) causes significant upper and lower respiratory disease in all age groups worldwide. However, there is no licensed drugs or vaccine available against hMPV. gamma-Fagarine, an alkaloid isolated from the root of zanthoxylum, has been reported to be effective in the treatment of cancer, inflammatory diseases and antivirals. However, little is known about the inhibitory effect of gamma-Fagarine against respiratory virus infection and the mechanism. In this study, we aim to investigate the effect of gamma-Fagarine on hMPV infection and explore its underlying molecular mechanisms. Vero-E6 and 16HBE cells were used as cell models. Virus replication and microcosm character were explored in Vero-E6 cells. Then, the antiviral activities were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), and indirect immunofluorescence assays (IFAs) in Vero-E6 and 16HBE. Potential mechanisms of gamma-Fagarine related to HSPG and lysosome pH were assessed in 16HBE cells. Lastly, a virus-infected mouse model was established and antiviral assay in vivo was conducted. gamma-Fagarine showed no toxicity toward Vero-E6 cells and 16HBE cells but demonstrated anti-hMPV activity. Virus titers of gamma-Fagarine group were reduced to 33% and 45% of the hMPV groups, respectively. Besides, mechanistic studies revealed that gamma-Fagarine could inhibit hMPV by dual mechanisms of direct restraining virus binding with HSPG and influencing lysosome pH. Furthermore, oral delivery of gamma-Fagarine to hMPV-infected mice at a dosage of 25 mg/kg reduced the hMPV load in lung tissues. After gamma-Fagarine treatment, pathological damage caused by viral infection was also ameliorated. These findings suggest that gamma-Fagarine has antiviral effects in vitro and in vivo, which are associated with its ability to restrain virus binding with HSPG and influence lysosome pH, thus indicating that gamma-Fagarine has the potential to serve as a candidate to fight against hMPV infection and other respiratory viruses such as influenza viruses and SARS-CoV-2. CI - Copyright (c) 2023 The Authors. Published by Elsevier B.V. All rights reserved.