Objective To study the protective effect of trigonelline on testicular injury induced by heat stress inmice. Methods A total of 120 ICR male mice were randomly divided into a control group, model group, and trigonellinegroups (25 mg/ kg, 50 mg/ kg, 100 mg/ kg), with 24 rats in each group. The lower abdomen of the mice in the model andtrigonelline groups was placed in a constant temperature water bath at 43℃ for 15 min to induce heat stress, and then thetrigonelline group was treated using trigonelline through intragastric gavage for 14 days. The mice in the control group andthe model group were treated using saline for 14 days. Animals were sacrificed at 1, 7, 10, and 14 days after heatstimulation. The genital organ index, a sperm count, and HE staining were used to observe changes in testicular tissuestructure and for a comparative analysis. Results Compared with the control group, the genital organ coefficient and thenumber of epididymal sperm in the model group were significantly decreased ( P < 0. 05). A histopathological analysisshowed obvious testicular injury in the mice. Trigonelline treatment increased the genital organ coefficient and the number ofspermatozoa in the epididymis, compared with the model group ( P < 0. 05). Histopathology showed that trigonellinetreatment significantly reduced heat stress-induced injury in the mice. Conclusions Trigonelline can significantly improvethe testicular coefficient and reduce heat stress-induced damage in testis tissue in mice, and it has a certain antagonistic effect.