Citrulline is an amino acid and abundant in watermelon, which can be utilized by some strains of lactic acid bacteria (LAB). Lactobacillus helveticus is one of the LAB that utilizes citrulline by arginine deiminase (ADI) pathway. Fermented milk is one of the daily products used for contributing the beneficial effects of LAB. This study examined the protective effects of citrulline enriched Lactobacillus helveticus ASCC 511 (LH511)-fermented milk (LHFM) on intestinal epithelial barrier function and inflammatory response in IPEC-J2 cells caused by pathogenic Escherichia coli. The effects of citrulline enriched fermented milk with LH511 (5%LHFM_Cit-4mM) was determined by MTT assay. Adhesion assay was performed to determine the anti-adhesion effects against enterohemorrhagic (EHEC) and entero-invasive (EIEC) Escherichia coli in IPEC-J2 cells. The cell integrity under the challenge of lipopolysaccharide (LPS) from E. coli O55:B5 was examined by transepithelial electrical resistance (TEER), qPCR and Western blot assay. Five percent of 4 mM citrulline in fermented milks with LH511 (5%LHFM_Cit-4mM) significantly stimulated the population of IPEC-J2 cells by 36%. Adhesion level of LH511 was significantly increased by 9.2% when incubated with 5%LHFM_Cit-4mM and 5%LHFM_Cit-4mM reduced the adhesion of EHEC and EIEC in IPEC-J2 cells by 35.79% and 42.74%, respectively. Treatment with 5%LHFM_Cit-4mM ameliorated LPS induced activated inflammatory cytokines expression (TNF-α, IL-6 and IL-8) and concentration (IL-6 and IL-8) and early apoptosis. And it restored the TEER, and regulated the expression of tight junction (TJ) proteins (zonula occluden-1 (ZO-1), occludin and claudin-1), toll-like receptors (TLRs) (TLR2 and TLR4) and negative regulators of TLRs signaling pathway (A20 and IRAK-M). In conclusion, our findings suggested that 5%LHFM_Cit-4mM had positive effects on improving and maintaining the intestinal epithelial cell integrity and inflammatory response under both normal and pathogenic LPS-stimulated conditions.