Citrulline is an amino acid and a precursor of arginine that is believed to have the same beneficial effects as arginine. Lactobacillus helveticus ASCC 511 (LH511) utilizes arginine to produce extra energy for cell growth via arginine deiminase (ADI) pathway. Supplementation of citrulline and LH511 is considered to provide both benefits. The effects of LH511+Cit-2mM were examined on IPEC-J2 cell line and were used to determine anti-adhesion effects against pathogenic infection, the effect on cell integrity by measuring transepithelial electrical resistance (TEER) and that on tight junction (TJ) proteins (claudin-1, occludin and zonula occluden-1 (ZO-1)) expression by qPCR and Western blot analysis. The anti-apoptotic effect was also determined by a flow cytometric method. The adhesion level of LH511 on IPEC-J2 cells was enhanced when incubated with 2 mM citrulline. LH511+Cit-2mM exhibited the protective effect against the adhesion of enterohemorrhagic (EHEC) and enteroinvasive (EIEC) Escherichia coli (E. coli), and it also significantly improved TEER and stimulated TJ proteins expression. Treatment with LH511+Cit-2mM showed greater effects than arginine and citrulline alone. This study suggests that LH511 enriched with citrulline might be a potential supplement for enhancing the health of the intestine.