Inflammatory responses triggered by activation of the lipopolysaccharide (LPS)/Toll-like receptor (TLR) 4 signaling pathway are a key mechanism in nonsteroidal anti-inflammatory drug-induced enteropathy. The aim of this study was to investigate the probiotic effect of Lactobacillus casei strain Shirota (LcS) on indomethacin-induced small intestinal injury. Rats pretreated with viable LcS or heat-killed LcS once or once daily for a week were administered indomethacin by gavage to induce injury. Anti-inflammatory effects of L-lactic acid (1-15 mM) were evaluated in vitro by use of THP-1 cells. One-week treatment with viable LcS prevented indomethacin-induced intestinal injury with increase in the concentration of lactic acid in small intestinal content and inhibited increases in myeloperoxidase activity and expression of mRNA for tumor necrosis factor-alpha (TNF-alpha) while affecting neither TLR4 expression nor the number of gram-negative bacteria in intestinal content, whereas neither heat-killed LcS nor a single dose of viable LcS inhibited intestinal injury. Prevention of this injury was also observed in rats given l-lactic acid in drinking water. Both L-lactic acid and LcS culture supernatant containing 10 mM lactic acid inhibited NF-kappaB activation and increases in TNF-alpha mRNA expression and TNF-alpha protein secretion in THP-1 cells treated with LPS. Western blot analyses showed that both L-lactic acid and LcS culture supernatants suppressed phosphorylation and degradation of I-kappaB-alpha induced by LPS without affecting expression of TLR4. These findings suggest that LcS exhibits a prophylactic effect on indomethacin-induced enteropathy by suppressing the LPS/TLR4 signaling pathway and that this probiotic effect of LcS may be mediated by L-lactic acid.