Lactic acid bacteria (LAB) are probiotics that provide numerous beneficial effects on the host body, especially on the intestine. Combining several strains of LAB, we prepared a formulation containing four different LAB and studied its anti-inflammatory activity both in vitro and in vivo. The formulation significantly reduced NO production from RAW 264.7 cells treated with bacterial lipopolysaccharide, indicating that the formulation might include anti-inflammatory activity. The formulation also suppressed inflammatory change induced by trinitrobenzene sulfonic acid (TNBS) in mice, where oral or rectal administration of the formulation protected the colon tissue from the damage by TNBS. Expressions of the IL-6 and FasL genes appeared to be down-regulated by the formulation in TNBS-treated colon tissues, suggesting that the suppression of those genes may be involved in the anti-inflammatory activity of the formulation.