Despite mounting evidence for dietary protease benefits, the mechanisms beyond enhanced protein degradation are poorly understood. This study aims to thoroughly investigate the impact of protease addition on the growth performance, intestinal function, and microbial composition of weaned piglets. Ninety 28-day-old weaned pigs were randomly assigned to the following three experimental diets based on their initial body weight for a 28-day experiment: (1) control (CC), a basic diet with composite enzymes without protease; (2) negative control (NC), a diet with no enzymes; and (3) dietary protease (PR), a control diet with protease. The results show that dietary proteases significantly enhanced growth performance and boosted antioxidant capacity, increasing the total antioxidant capacity (T-AOC) levels (p p p p 0.01), and inhibited MAPK and NF-κB signaling pathways. Moreover, protease-supplemented diets improved intestinal morphology and barrier integrity, including zonula occludens protein 1(ZO-1), Occludin, and Claudin-1 (p Lachnospiraceae_AC2044_group and Prevotellaceae_UCG-001) (p Terrisporobacter (p < 0.05). Further correlation analysis revealed a positive link between beneficial bacteria and growth performance and a negative association with inflammatory factors and intestinal permeability. In summary, dietary protease addition enhanced growth performance in weaned piglets, beneficial effects which were associated with improved intestinal barrier integrity, immunological response, and microbiota composition.
