BACKGROUND: Microbiota and tight junction proteins (TJPs) are components of the gut barrier, and are considered stress targets that have deleterious effects on intestinal homeostasis. OBJECTIVES: This study aimed to evaluate the effects of chronic immobilization stress on selected small intestine homeostasis parameters. MATERIAL AND METHODS: Female BALB/c mice were divided into a stress group that underwent short-term immobilization for 2 h per day for 4 consecutive days, and a non-stressed control group (n = 6 per group). Proximal and distal small intestine samples were excised to assess colony-forming units per gram (CFU/g) of total bifidobacteria in selective agar plates, luminal albumin was assessed using immune-enzymatic assay, pro-inflammatory cytokines were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and TJPs (pore-forming, claudin (Cld)-2; pore-sealing, Cld-4; ambiguous, Cld-7, -12 and -15) were assessed with RT-qPCR and western blotting. RESULTS: Compared with the control group, the stress group had lower body weight and energy intake. In the distal region, the stressed mice had lower bifidobacteria count and messenger ribonucleic acid (mRNA) expression of Cld-2, Cld-4 and Cld-12, though they had more albumin and higher interleukin (IL)-6 mRNA expression. Within the proximal region, the stressed mice had higher mRNA expression of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-6, Cld-7, Cld-12, and Cld-15, along with lower levels of IL-10 and Cld-4. However, mRNA and protein expression of TJPs were discordant. CONCLUSIONS: These findings indicate divergent stress-induced outcomes in the small intestine, evidenced by the elicitation of a pro-inflammatory response and decreased anti-inflammatory response in the duodenum, and by increased albumin transudation and decreased bifidobacterial growth in the distal region.