Developing effective strategies to prevent diarrhea and associated-gut disorders in mammals has gained great significance. Owing to the many health benefits provided by the commensal microbiota of the intestinal tract, such as against environmental perturbation, we explored the host phenotype-associated microbes and their probiotic potential. Based on the observations that the chronic heat stress-exposed weaned piglets present as heat stress-susceptible (HS-SUS) or heat stress-resistant (HS-RES) individuals, we confirmed the phenotypic difference between the two on growth performance (P < 0.05), diarrhea index (P < 0.001), intestinal heat shock protein 70 (HSP70) regulation (P < 0.01), and inflammatory responses (P < 0.01). By comparing the gut microbiome using 16S rRNA gene sequencing and KEGG functional analysis, we found that Lactobacillus johnsonii exhibited significantly higher relative abundance in the HS-RES piglets than in the HS-SUS ones (P < 0.05). Further experiments using a mouse model for chemical-induced inflammation and intestinal injury demonstrated that oral administration of a representative L. johnsonii N5 (isolated from the HS-RES piglets) ameliorated the clinical and histological signs of colitis while suppressing intestinal pro-inflammatory cytokines TNF-α and IL-6 production (P < 0.05). We found that N5 treatment enhanced tight junction proteins ZO-1 and occludin and cytoprotective HSP70 levels under physiological condition and restored their mucosal expressions in colitis (P < 0.05). In support of the high production of the anti-inflammatory cytokine IL-10, N5 promoted the intestinal Peyer's patches MHCII+ and CD103+ dendritic cell populations (P < 0.05), increased the regulatory T (Treg) cell numbers (P < 0.05), and decreased the Th17 population and its IL-17a production under physiological condition and during colitis (P < 0.01). Our results shed light on understanding the interaction between commensal Lactobacillus and the host health, and provide L. johnsonii N5 as an alternative to antibiotics for preventing diarrhea and intestinal diseases.