Heat stress (HS) can lead to oxidative stress and inflammatory responses, which jeopards animal production. Enzymatically treated mulberry leaf protein (ETMLP) has antioxidant activities and immune regulation. This study aimed to investigate the impacts of ETMLP on mitigate liver injury caused by heat stress in animals. All 25 rats were divided into five groups: control, heat stress, and three ETMLP supplementation groups (HS + 0.4 %, HS + 0.8 %, and HS + 1.2 %). ETMLP supplementation effectively reduced markers of liver damage and increasing the level of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-px). It also regulated protein expression associated with stress response and antioxidant defense and suppressed pro-inflammatory proteins and pathways expression. Moreover, ETMLP improved intestinal morphology and increased the intestinal barrier gene abundance. ETMLP also positively influenced the intestinal microbiota by increasing beneficial microorganisms. Overall, ETMLP reversed heat stress-induced damage, improved growth performance, and regulated the intestinal microbiota in the study animals.