In Salmonella Typhimurium (S. Typhimurium), lipopolysaccharide (LPS) anchored on the bacterial outer membrane is a major immune stimulus that can broadly activate immune cells and induce innate immune responses. wzxE is involved in bacterial LPS biosynthesis but has rarely been reported in Salmonella; wzxE encodes a flipase that can flip the precursor of LPS across the membrane into the periplasm space. Our preliminary data showed that the wzxE transposon mutant of S. Typhimurium could not significantly adhere to and invade into HEp-2 cells, but the mechanism remains unknown. In this study, we infected human LS174 T, Caco-2, HeLa, and THP-1 cells with the wild-type S. Typhimurium strain SL1344, its wzxE mutant, and its complemented strain. wzxE depletion significantly attenuated bacterial adhesion and internalization in the four cell types. In addition, the postinfectious production of interleukin-8 (IL-8) was significantly decreased in the Caco-2 cells infected with the wzxE mutant. Bacterial LPS stained with polymyxin B probe also exhibited a reduced signal in the wzxE mutant. The silver staining of purified LPS demonstrated a significant reduction of the O-antigen (OAg) chain in the wzxE mutant. To confirm the role of OAg in the wzxE mutant during infection, we treated the HT-29 cells with the S. Typhimurium strain SL1344, its wzxE mutant, and their purified LPS, which revealed significantly decreased IL-8 secretion in the HT-29 cells treated with purified LPS from the wzxE mutant and with the wzxE mutant. In conclusion, wzxE mediates LPS biosynthesis and plays a major role in bacterial pathogenesis by regulating OAg flipping.