To verify the related risk factors of crayfish-related rhabdomyolysis outbreak using animal experiment, and to provide positive samples by animal experiment screening for the confirmation of the pathogenic factors of crayfish-related rhabdomyolysis. Methods Based on the result of epidemiological investigations and the traceability information of 48 crayfish-related rhabdomyolysis cases in Guangdong Province in 2016, suspected crayfish samples were screened and separated as meat and hepatopancreas, which were given to mice by gavage to trigger crayfish-related rhabdomyolysis through observing the indicators of serology and skeleton histopathology. Results The main clinical symptoms of the cases were muscle soreness, fatigue, dyspnea and chest distress, and the blood biochemistry test showed that the abnormal percentage of creatine kinase(CK), creatine kinase isoenzyme(CKMB)and myoglobin(MB)were above 85%, accordance with the diagnosis of haff disease. The crayfish-related rhabdomyolysis was related with the batch and intake of crayfish, as well as the individual physical difference. In 36 incidents, there were two restaurants (restaurant A and restaurant B) reported 2 incidents respectively, others only reported one. 30 g/kg BW of female hepatopancreas from restaurant A could induce increase of serum CK and CKMB in mice. After giving the highest dose (30 g/kg BW) of hepatopancreas from restaurant A, serum CK and CKMB were all higher than other dose groups (10,3.3,1.1 g/kg BW). 30 g/kg BW crayfish meat from restaurant A and B could both induce increase of serum CKMB, MB, aspartate aminotransferase(AST), lactate dehydrogenase(LDH)and creatinine(CR). The abnormal increase of rhabdomyolysis related indicators CK, CKMB, MB, LDH, glutamic pyruvic transaminase(ALT)and AST were positively correlated in the same animal. The rhabdomyolysis pathological manifestations, such as inflammatory cell infiltration, eosinophilic in bicepsfemoris muscle, and cross striations unclear and disappear were mainly observed in animals with serum CK, CKMB abnormal increase. Conclusion Crayfish-related rhabdomyolysis could be triggered in mice, and was associated with the batch and dose of the crayfish, which was consistent with the result of human epidemiological investigation.