Abstract: To investigate the digestive characteristics of milk and soybean powders from different sources in the infant gastrointestinal tract and their effects on gut microbiota, this study compared cow milk powder (CM), goat milk powder (GM), sheep milk powder (SM), and soybean powder (SB). An in vitro continuous infant gastrointestinal digestion model combined with fecal fermentation was established to evaluate protein hydrolysis, short-chain fatty acid (SCFA) production, and gut microbial composition. Results showed GM and CM exhibited significantly higher in vitro protein hydrolysis rates than SM and SB (P<0.05). During in vitro fecal fermentation, the three animal milk powders increased butyric acid and valeric acid concentrations by 4~7 times and 1~2 times, respectively. Microbiota analysis revealed that CM and SM generated gut microbial profiles more similar to those of breastfed infants, significantly increasing the abundance of Anaerococcus (P<0.05), which was associated with butyrate production. In contrast, SB showed lower protein digestibility, significantly elevated acetic acid levels and disrupted microbial homeostasis. It was characterized by a marked reduction in the abundance of beneficial genera such as Bifidobacterium and Clostridium_sensu_stricto_1. Correlation analysis further confirmed a strong negative correlation between acetic acid concentration and Bifidobacterium abundance (P<0.01). In summary, goat-milk powder and sheep-milk powder were more suitable raw materials for infant formula, offering scientific evidence to inform the rational selection of base powders in infant formula development.