Helicobacter pylori infection is thought to be involved in the development of several gastric diseases. Two H. pylori virulence factors (vacuolating cytotoxin A and cytotoxin-associated gene A) reportedly interact with lipid rafts in gastric epithelial cells. The role of Toll-like receptor (TLR)-mediated signaling in response to H. pylori infection has been investigated extensively in host cells. However, the receptor molecules in lipid rafts that are involved in H. pylori-induced innate sensing have not been well characterized. This study investigated whether lipid rafts play a role in H. pylori-induced ceramide secretion and TLR4 expression and thereby contribute to inflammation in gastric epithelial cells. We observed that both TLR4 and MD-2 mRNA and protein levels were significantly higher in H. pylori-infected AGS cells than in mock-infected cells. Moreover, significantly more TLR4 protein was detected in detergent-resistant membranes extracted from H. pylori-infected AGS cells than in those extracted from mock-infected cells. However, this effect was attenuated by the treatment of cells with cholesterol-usurping agents, suggesting that H. pylori-induced TLR4 signaling is dependent on cholesterol-rich microdomains. Similarly, the level of cellular ceramide was elevated and ceramide was translocated into lipid rafts after H. pylori infection, leading to interleukin-8 (IL-8) production. Using the sphingomyelinase inhibitor imipramine, we observed that H. pylori-induced TLR4 expression was ceramide dependent. These results indicate the mobilization of ceramide and TLR4 into lipid rafts by H. pylori infection in response to inflammation in gastric epithelial cells.