This study investigated the in vitro antibacterial activity of Lactobacillus acidophilus AD125 against Escherichia coli (E. coli) O157:H7 and its probiotic properties: gastrointestinal tolerance, surface hydrophobicity, autoaggregation, coaggregation, and adhesion to Caco-2 cells. In addition, the action mode of the strain's antagonism against adhesion of E. coli O157:H7 to Caco-2 cells was analyzed, and related substances were also determined. Results showed that L. acidophilus AD125 had stronger antibacterial activity (inhibition zone of 20.47 ± 0.43 for AD125 culture solution and 14.55 ± 1.12 for cell-free supernatant) against E. coli O157:H7 than other Lactobacillus spp. Also, this strain had higher gastrointestinal tolerance, autoaggregation percentage (26.51 ± 0.71%), and coaggregation percentage (23.97 ± 0.44%) with E. coli O157:H7. High surface hydrophobicity of toluene and xylene (83.59 ± 2.54% and 93.45 ± 1.24%) was also observed. Bacterial adhesion counts were 1176.54 100 per cells, indicating good adhesion to Caco-2 cells. Furthermore, the exclusion, competition, and antibacterial effect of AD125 may have driven its antagonism against E. coli O157:H7 adhesion. Finally, surface-layer proteins, extracellular polysaccharides, and thermosensitive substances all participated in the antagonism against E. coli O157:H7, with surface-layer proteins the main related substances. These results show that Lactobacillus acidophilus AD125 is promising for inhibiting E. coli O157:H7 and preventing and treating intestinal diseases induced by E. coli O157:H7.