The aim of the present study was designed to investigate that Platycodon grandiflorum saponins (PGS) exerted protective effects against testicular injury induced by scrotal hyperthermia. PGS (15, 30 mg/kg) were administered intragastrically to mice for 14 days, then exposed to a single scrotal heat treatment at 43 °C for 18 min on the 7th day. Heat stress (HS) induced spermatogenic damage was associated with a significant loss of leydig cells and the changes of serum testosterone, oxidative stress and apoptosis markers, and activated MAPK signaling pathway. In contrast, PGS treatment normalized HS-induced histopathological changes and prevented the activation of MAPK signaling pathway, which was contributed to oxidative stress and apoptosis in the testis of treated mice. In conclusion, our findings clearly demonstrated that PGS exhibited a significant protective effect against HS-induced testicular dysfunction, largely dependent upon the inhibition of oxidative stress associated apoptosis via partly regulation of MAPK signaling pathway.