〔Abstract〕 Objective To investigate the effect of isoacteoside on the replication process of respiratory syncytial virus (RSV) and its underlying mechanism. Methods RSV-infected HEp-2 cells (control group) were treated with isoacteoside (isoacteoside-treated group). Real-time quantitative PCR (qPCR) was used to analyze the mRNA expression levels of the RSV fusion protein (F) gene and matrix protein (M) gene in both groups. Viral titers were determined by plaque assay to assess the inhibitory effect of isoacteoside on viral replication. The number of RSV inclusion bodies formed was determined using cell transfection and laser confocal imaging to evaluate the role of isoacteoside in the formation of inclusion bodies required for RSV replication. 18 seven-week-old female BALB/c mice were randomly divided into three groups: uninfected control group (n = 6), RSV-infected group (n = 6), and RSV-infected group treated with isoacteoside (n = 6). 5 days after infection, the mice were euthanized and lung tissues were collected for pathological analysis to assess the therapeutic effects of isoacteoside on RSV-induced lung tissue injury. Results Compared to the control group, the isoacteoside-treated group exhibited the reduced mRNA levels ofthe RSV F gene (t = 17.13, P < 0.001) and M gene (t = 18.22, P < 0.001), as well as a decrease in viral titer (t = 15.32, P < 0.001). The number of inclusion bodies was also significantly reduced (t = 16.12, P < 0.001). In the RSV-infected mouse experiment, compared to the RSV-infected group, the isoacteoside-treated group showed a decrease in the levels of inflammatory cytokines IL-4 (t = 14.76, P < 0.01) and IL-6 (t = 21.13, P < 0.001) in lung tissues, as well as decreased mRNA levels of the viral load–associated genes F (t = 19.52, P < 0.001) and M (t = 18.76, P < 0.001) in lung tissues. Pathological damage to lung tissue caused by RSV infection was alleviated. Conclusion Isoacteoside inhibits RSV replication by interfering with the formation of RSV inclusion bodies and alleviates lung tissue injury induced by RSV infection in mice.