Abstract: Rapamycin is a macrolide antibiotic agent, which is effective for many kinds of human diseases. Recently, it has been found that rapamycin has good antibacterial activity against a variety of plant pathogenic fungi. It is expected to become a microbial pesticide for plant diseases. However, there are no reports about the hydrolysis behavior and its acute toxicity to non-target organisms of rapamycin. In order to evaluate the hydrolysis behavior of rapamycin and determine its acute toxicity to non-target organisms, the hydrolysis properties of rapamycin under different pH values, temperatures initial concentrations, and light sources were studied by the indoor simulation test method. Besides, the acute toxicity of rapamycin to four non-target organisms, such as Bombyx mori, Apis melliifera L., Brachydanio rerio and Eisenia foetida were determined. The results showed that alkaline environment,ultraviolet light and high temperature were conducive to the hydrolysis of rapamycin. When the initial mass concentration was 10 mg/L, the hydrolysis half-lives were 66.63, 38.93 and 77.00 h respectively. When the initial mass concentration was 50 mg/L, the hydrolysis half-lives were 144.38, 105.00 and 165.00 h respectively. Acute toxicity test showed that the 96 h-LC₅₀ active ingredients values of rapamycin to B. mori and B. rerio were 386.46 and 3.50 mg a.i./L, respectively. The 14 d-LC₅₀ active ingredients value for E. foetida was 223.81 mg a.i./kg. The acute oral toxicity of 48 h-LD₅₀ active ingredients value to A. melliifera L. was 8.95 μg a.i./bee and acute contact toxicity was 16.79 μg a.i./bee. According to the test guidelines on environmental safety assessment for chemical pesticides (GB/T 31270—2014), The acute toxicity of rapamycin to B. mori, B. rerio, and E. foetida were low toxicity, medium toxicity, and low toxicity respectively. The acute oral toxicity and contact toxicity to A. melliifera L. were medium toxicity and low toxicity respectively. The results showed that rapamycin was a biodegradable agent and was safe to four non-target organisms including: B. mori, A. melliifera L., B. rerio and E. foetida.