We have previously shown that non-pathogenic Gram-negative Bacteroides vulgatus induces transient RelA phosphorylation (Ser-536), NF-kappaB activity, and pro-inflammatory gene expression in native and intestinal epithelial cell (IEC) lines. We now demonstrate that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) but not prostaglandin E(2) inhibits lipopolysaccharide (LPS) (B. vulgatus)/LPS (Escherichia coli)-induced RelA phosphorylation and interleukin-6 gene expression in the colonic epithelial cell line CMT-93. This inhibitory effect of 15d-PGJ(2) was mediated independently of LPS-induced IkappaBalpha phosphorylation/degradation and RelA nuclear translocation as well as RelA DNA binding activity. Interestingly, although B. vulgatus induced nuclear expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in native epithelium of monoassociated Fisher rats, PPARgamma-specific knock-down in CMT-93 cells using small interference RNA failed to reverse the inhibitory effects of PPARgamma agonist 15d-PGJ(2), suggesting PPARgamma-independent mechanisms. In addition, 15d-PGJ(2) but not the synthetic high affinity PPARgamma ligand rosiglitazone triggered ERK1/2 phosphorylation in IEC, and most importantly, MEK1 inhibitor PD98059 reversed the inhibitory effect of 15dPGJ(2) on LPS-induced RelA phosphorylation and interleukin-6 gene expression. Calyculin A, a specific phosphoserine/phospho-threonine phosphatase inhibitor increased the basal phosphorylation of RelA and reversed the inhibitory effect of 15d-PGJ(2) on LPS-induced RelA phosphorylation. We further demonstrated in co-immunoprecipitation experiments that 15d-PGJ(2) triggered protein phosphatase 2A activity, which directly dephosphorylated RelA in LPS-stimulated CMT-93 cells. We concluded that 15d-PGJ(2) may help to control NF-kappaB signaling and normal intestinal homeostasis to the enteric microflora by modulating RelA phosphorylation in IEC through altered protein phosphatase 2A activity.