Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and represents one of the most critical microbe-associated molecular pattern (MAMP) molecules. In this study, we isolated and purified LTA from Clostridium butyricum (bLTA) and compared its effects on the inflammatory responses of HT-29 cells with those of LTA from Staphylococcus aureus (aLTA). We also compared the effects of bLTA and aLTA on cell growth, proliferation, and apoptosis. The results showed that the length and saturation degree of the acyl chains in the two LTA molecules were obviously different. aLTA stimulated the phosphorylation of p65 and activated the NF-κB signaling pathway, inducing the expression and secretion of cytokines. Moreover, aLTA also inhibited the growth and proliferation of HT-29 cells and induced cell apoptosis. However, bLTA had no significant effects on the NF-κB signaling pathway in HT-29 cells and did not stimulate cellular inflammatory responses or induce apoptosis. These differences in activity may result from the different lengths and saturation degrees of the acyl fatty acid chains of the two LTA molecules. These differences may also account for the distinct effects elicited by probiotic bacteria and pathogenic bacteria on host cells.