The viability of most probiotics is generally reduced by gastric and bile acids during passage through the host's gastrointestinal tract. Therefore, we investigated the effect of encapsulation with poly-γ-glutamic acid (γ-PGA) on the viability and function of probiotics. The protective effect of γ-PGA encapsulation at different γ-PGA concentrations (0.25, 0.5, 0.75, and 1.0%) and simulated gastric juice (SGJ) exposure times (1, 2, and 3 h) was assessed. The viability of all probiotics was either completely lost or significantly reduced within 3 h of exposure to SGJ under non-encapsulation conditions. In contrast, γ-PGA encapsulation at a concentration of 0.25–1.00% maintained the viability of all SGJ-exposed strains. In an in vitro gastrointestinal digestion experiment simulating the oral, gastric, and duodenal phases, encapsulation resulted in a higher survival rate than that associated with non-encapsulation. In vivo experiments involving oral administration of encapsulated Limosilactobacillus reuteri to mice showed that encapsulation increased the abundance of the genus Lactobacillus in the gut microbiota and increased propionic acid content among the short-chain fatty acids (SCFAs) analyzed. In addition, live probiotic cell concentrations in mouse feces were found to be significantly higher in the encapsulated probiotic-fed group than in the non-encapsulation group. The present findings demonstrate that γ-PGA encapsulation maintains the viability of probiotics in the gastrointestinal tract of the host, while providing beneficial effects on changes in the gut microbiota and SCFA content.