Lactobacillus salivarius is a species of lactic acid bacteria with probiotic potency. Compared to such well-known probiotics as L. rhamnosus and L. casei, the genomic characteristics and health-beneficial effects of L. salivarius are inadequately researched. For this study, a medium with enhanced selectivity for the isolation of L. salivarius was developed by optimizing the carbon source and antibiotics in the medium. Seventy-three L. salivarius strains were isolated from 472 fecal samples from Chinese populations, and their pan-genomic and phylogenetic characterizations were analyzed. Three strains (L. salivarius HN26-4, NT4-8, and FXJCJ7-2) that were clearly categorized in different sub-phylotypes of the phylogenetic tree were randomly selected for further studies. Compared to the other two tested strains, L. salivarius FXJCJ7-2 showed higher tolerance to simulated gastrointestinal tract conditions and more significant anti-inflammatory effects in lipopolysaccharides (LPS)-treated RAW264.7 murine macrophages. This strain was also more effective in reversing LPS-induced alterations in gut barrier function, colonic histopathology, Treg/Th-17 balance, immunomodulatory indicators, nuclear factor kappa B pathway activation, and the intestinal microenvironment of the mice than the other two tested strains. Comparative genomic analysis indicated that these protective effects may be related to the specific genes of L. salivarius FXJCJ7-2 that were involved in the tolerance to the gastrointestinal environment, short-chain fatty acid production, and host-bacterium interaction.