Abstract: In vitro experiments were conducted to investigate the effects of concentration, growth stage and surface layer protein of Lactobacillus acidophilus KLDS1.0901 on its ability to adhere to Caco-2 cells. The untreated KLDS1.0901, the remove surface layer protein and the heat-killing KLDS1.0901 were fluorescently labeled with cFDA-SE, then administered to mice, and the intestinal mucosa of different parts of the intestine was taken for flow cytometry. Surface layer protein of KLDS1.0901 was crudely extracted by 5 mol/L LiCl and identified by transmission electron microscopy and SDS-PAGE. The results showed that with the increase of bacterial concentration, the adhesion rate of KLDS1.0901 increased, but reached saturation when the concentration was 10⁸ CFU/mL. At the end of the logarithmic growth phase, the bacteria had better adhesion properties. After the Caco-2 monolayer was co-incubated with the surface layer protein for 1 h, the adhesion amount of KLDS1.0901 to it was significantly decreased. And the higher the concentration of the surface layer protein added, the stronger the inhibition of adhesion. Under transmission electron microscopy, the untreated KLDS1.0901 cell wall was surrounded by a surface structure, which disappeared after LiCl treatment. The extracted crude protein had a surface layer protein content of approximately 15.6% and a molecular weight of approximately 43 kDa. The results showed that Lactobacillus acidophilus KLDS1.0901 was rich in surface layer protein and could adhere to the intestinal tract of mice well. It had the potential of probiotics.