Abstract: Rapamycin is an important marocyclic polyketide produced by Streptomyces hygroscopicus, which exhibited immunosuppressive and antifungal properties. In this study, antifungal activity of rapamycin against four kinds of plant pathogens, including Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, and Fusarium oxysporum, was assayed by the mycelium growth rate method. The results showed that rapamycin exhibited extreme significant inhibit efficacy against mycelial growth of all of these plant pahogens on the PDA medium. The EC50 value of rapamycin against S. sclerotiorum was 2.23 × 10-4 μg/mL, which is the lowest. The EC50 values against B. cinerea, R. solani, and F. oxysporum were 1.32 × 10-3, 4.05 × 10-3 and 3.82 × 10-3 μg/mL, respectively. It is noteworthy that the toxicity of rapamycin against the mycelial growth of B. cinerea is significantly higher than those of azoxystrobin and propamidine, with the EC50 values of 3.24 and 3.81 μg/mL, respectively. The hypha of B. cinerea exhibited premature aging after treated with rapamycin under the scanning electron microscope. The results in this study provided the basis for investigating the efficient inhibitation mechanism of rapamycin in the future study.