Objective The acute toxicity and rectal mucosal irritation of Reduning Injection enema were studied in order to provide reference for clinical safety of Reduning enema. Methods Forty juvenile rats were randomly divided into acute toxicity control group and experimental group, rectal mucosal stimulation control group and experimental group. The acute toxicity group was given rectal administration of 20 mL/kg each time, repeated three times at an interval of 2 hours, and the control group was given an equal amount of 0.9% normal saline enema. The general condition, weight and number of dead juvenile rats were observed after 7 days. The rectal mucosal stimulation group was given rectal administration of 2 mL/kg per day, according to the clinical dosage, and the rectal mucosal stimulation was observed after continuous administration for 7 days. Results There was no significant difference in body mass between the acute toxicity experimental group and the control group after enema (P＞0.05). There were no poisoning symptoms and no death of the juvenile rats 7 days after enema.Rectal mucosa irritation of juvenile rats in the experimental group was similar to that in the control group. No injury or secretion was observed in the mucosa, and some mild to moderate hyperemia was observed. Conclusion The rectal administration of Reduning Injection is safe and non-toxic, with mild irritation to the rectal mucosa without damage.