Intestinal alkaline phosphatase (IAP) is an intestinal brush border enzyme which plays an important role in gut homeostasis and mucosal inflammation. However, the mechanism of the protective effect of IAP is not fully elucidated. The aim of the present study was to evaluate whether the protective effect of IAP on colitis is mediated via the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway. For in vitro analysis, peritoneal macrophages from the wild-type (WT) and TLR4-deficient (TLR4-/-) C57BL/6 mice were used. IAP strongly inhibited the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) by suppressing the LPS-induced phosphorylation of IκBα and the DNA-binding activity of NF-κB in WT macrophages; however, the inhibitory effects mediated by IAP were reduced in the TLR4-/- macrophages. For in vivo analysis, the protective mechanisms of IAP on dextran sulfate sodium (DSS)-induced colitis were evaluated using WT and TLR4-/- mice. Although oral administration of IAP significantly attenuated the severity of colitis in both preventive and therapeutic models of WT mice, these protective effects were not significant in TLR4-/- mice. When immunohistochemical analysis of IκBα was performed in the colitic tissues, the inhibitory effects of IAP on IκBα phosphorylation were also observed in the colon of WT mice, but these effects decreased in the colon of TLR4-/- mice. In conclusion, the protective effects of IAP on colitis were mediated via the TLR4/NF-κB pathway. These results of this study shall be helpful in tailoring treatment against colitis using IAP.