Objective To explore the effect of rifampin on yellow fever virus (YFV) and its preliminary mechanism. Methods Indicated concentrations of rifampin (0.04, 0.2, 1, 5, 25, 125, 625 μmol/L) were incubated with human liver cancer cells Huh-7 for 24 h. The cytotoxicity of rifampin was measured by CCK-8 assay and the 50% cytotoxic concentration (CC₅₀) was calculated. When Huh-7 cells were infected with YFV, different concentrations of rifampin (0.04, 0.2, 1, 5, 25 μmol/L) were added to detect the dose-response relationship of YFV resistance and to calculate the half inhibition concentration (IC₅₀); rifampin (5 μmol/L) was added and incubated for different exposure time (2, 4, 8, 12, 24 h) to detect the time-effect relationship of YFV resistance. Huh-7 cells were infected with YFV for 2 h, and rifampin (25 μmol/L) was added at different time points (2, 4, 6, 8, 12 h) for 2 h incubation. The most significant anti-YFV stage was detected. The effect of rifampin on YFV invasion was evaluated by virus binding assay and cholera toxin B (CTB)/transferrin (TF) mediated endocytosis experiment. Results Rifampin had a robust anti-YFV effect (IC₅₀=1.868 μmol/L, P<0.01) with low cytotoxicity (CC₅₀=176.9 μmol/L). Kinetic test, binding and endocytosis experiments showed that rifampin could significantly inhibit YFV binding and invading target cells (P<0.01), but did not affect the endocytosis process of YFV. Moreover, rifampin also had inhibitory effect at late YFV replication stage (P<0.01). Conclusion Rifampin can inhibit YFV infection by blocking viral binding to the target cells at early stage of viral entry.