Testicular hyperthermia induced by unhealthy living habits and pathological or occupational factors can cause spermatogenic dysfunction with an outcome of sub-fertility or even infertility. Cyanidin-3-O-glucoside (C3G) is the most typical anthocyanin in foods that has been recognized as an antioxidant with promising protection for male reproduction. However, its specific effect against testicular hyperthermia and the mechanisms involving its primary gastrointestinal metabolite protocatechuic acid (PCA) are still unexplored. In the present study, testicular hyperthermia in mice was established by employing a single hot water bath at 43 °C for 30 min. C3G and PCA were intragastrically given to investigate their prevention ability against heat stress-induced testicular damage. It was found that C3G and PCA restored the external diameter and thickness, and alleviated atrophy and vacuolation of seminiferous tubules. Simultaneously, C3G and PCA enhanced testicular heat stress tolerance through reducing superfluous eIF2α phosphorylation and stress granule formation. C3G and PCA effectively improved the testicular antioxidant system and regulated the IRE1α-XBP1 pathway, contributing to mitigatory spermatogenesis dysfunction and testicular damage. This finding revealed that anthocyanins were the novel compounds for alleviating testicular damage, and provided a reliable theoretical basis for improving male fertility disturbed by heat stress.