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We confirmed that Lactobacillus paracasei (L. paracasei) strains (KBL382, 384, 385) exhibited strong protective effects in an in vivo dextran sodium sulfate-induced colitis model. L. paracasei treatment significantly improved colitis symptoms. Among tested strains, L. paracasei KBL382-treated mice exhibited significantly lower levels of cytokines interferon gamma, interleukin (IL)-4, IL-6, tumor necrosis factor, and IL-17A. On the other hand, higher levels of anti-inflammatory cytokine IL-10 and CD4 + CD25 + Foxp3+ T regulatory cell populations in mesenteric lymph nodes were observed in mice group with L. paracasei. L. paracasei treatment clearly increased zonula occluden-1, claudin 3, and mucin 4 levels, which improved the tight junction and mucus thickness. L. paracasei-treated mice showed significantly higher bacterial diversity and changed the composition of cecal microbiota. Therefore, we suggest that L. paracasei could be a useful tool in the treatment of inflammatory bowel diseases via immunomodulation and the improvement of gut microbiota.