S-layer proteins in Lactic acid bacteria are not the only cell surface structures used for aggregation, but also plays significant role for intestinal tissue adhesion along with some other functional elements. In addition, it was determined that the properties of S-layer proteins differs not only between species but also the strains which belong to same species. In this work, presence and some functions of S-layer in lactic acid bacteria were determined, its effect on resistance to gastrointestinal enzymes, aggregation and adhesion ability were investigated as well. For this purpose S-layers of microorganisms were removed by 5 M LiCl treatment and size of the proteins were determined by SDS-PAGE analysis. The removal of S-layer proteins caused a change in the resistance of microorganisms to GIS enzymes. After the S-layer removal, two strains considerably lost their resistance to GIS enzymes. The strains mostly lost their aggregation ability in the absence of S-layer. The results showed that S-layer proteins are not the only structures involved in aggregation processes but, is a major mediator in Lactobacilli. Removal of S-layer had no effect on adhesion ability of W. cibaria DA28, the effect on L. casei DA4, L. coryniformis DA263 and L. plantarum DA140 was moderate, but the effect was high on L. plantarum DA100. The study showed that S-layer proteins play limited protection against GIS enzymes. In addition, absence of S-layer adversely affected aggregation and adhesion ability of strains.