The gut microbiota plays an important role in multifaceted physiological functions in the host. Previous studies have assessed the probiotic effects of Lactobacillus salivarius LI01. In this study, we aimed to investigate the potential effects and putative mechanism of L. salivarius LI01 in immune modulation and metabolic regulation through the monocolonization of germ-free (GF) Sprague-Dawley (SD) rats with L. salivarius LI01. The GF rats were separated into two groups and administered a gavage of L. salivarius LI01 or an equal amount of phosphate-buffered saline. The levels of serum biomarkers, such as interleukin (IL)-1 alpha, IL-5, and IL-10, were restored by L. salivarius LI01, which indicated the activation of Th0 cell differentiation toward immune homeostasis. L. salivarius LI01 also stimulated the immune response and metabolic process by altering transcriptional expression in the ileum and liver. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment of the 5 '-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, which indicated that L. salivarius LI01 exerts an effect on energy accumulation. The LI01 group showed alterations in fecal carbohydrates accompanied by an increased body weight gain. In addition, L. salivarius LI01 produced indole-3-lactic acid (ILA) and enhanced arginine metabolism by rebalancing the interconversion between arginine and proline. These findings provide evidence showing that L. salivarius LI01 can directly impact the host by modulating immunity and metabolism.