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<i>Lactobacillus</i> are normal inhabitants of the gastrointestinal tract and confer a variety of health effects. Lipoteichoic acid (LTA), an amphiphilic substance located in the cell membrane, is a key molecule in probiotic–host crosstalk. Through the characterization of structural characteristics of LTA molecules derived from <i>Lactobacillus plantarum</i> A3, <i>Lactobacillus reuteri</i> DMSZ 8533, and <i>Lactobacillus acidophilus</i> CICC 6074, there exists some heterogeneity in LTA molecules, which perhaps contributes to the distinguishable adhesion properties of <i>Lactobacillus</i> strains based on fluorescence microscopy observations. In LPS-induced RAW 264.7 cells, LTAs derived from three <i>Lactobacillus</i> strains obviously alleviated inflammatory responses as evidenced by the altered inflammatory cytokine levels of TNF-α, IL-6, and IL-10. Western blotting demonstrated that <i>L. reuteri</i> LTA blocked LPS-triggered expression of the MAPK and NF-κB pathways. The findings further validated that LTA is an important effector molecule and deserves further consideration as an alternative therapeutic for ulcerative colitis treatment.