An adhesion-related protein of Lactobacillus acidophilus strain CICC 6074 involved in binding to Caco-2 cells and inhibiting Enteropathogenic Escherichia coli (EPEC) was isolated and characterized. The S-layer protein was extracted with 5M LiCl and the active fraction purified by gel filtration (G-75). The S-layer protein was visualized by SDS-PAGE and characterized by estimating the relative molecular weight using mass spectra. The inhibitory effect of L. acidophilus and its S-layer proteins on the ability of EPEC to adhere to cells was explored by using a Caco-2 cell model. The results suggest that the S-layer proteins of L. acidophilus are adhesive in nature and are involved in the competitive exclusion of EPEC from Caco-2 cells.