The role of fibronectin binding protein A (FbpA) in Listeria monocytogenes infection and its pathogenesis were studied in vivo and in vitro by constructing a fbpA-deficient mutant of L. monocytogenes (ΔfbpA). In vivo, ΔfbpA was less pathogenic in mutant mice than was wild-type L. monocytogenes. FbpA did not affect the amounts of various virulence-determining factors, including internalin B and listeriolysin O. However, adherence to, and invasion of, mouse hepatocytes by the ΔfbpA mutant were reduced. In contrast, adherence to, but not invasion of, the ΔfbpA mutant to macrophages was attenuated. Fibronectin contributed to the efficient adherence and invasion of wild-type L. monocytogenes, but not to those of the ΔfbpA mutant. Attenuation of adhesion and uptake of the ΔfbpA mutant were reversed by overexpression of FbpA in it. FbpA was not involved in intracellular growth, autophagy induction or actin tail formation. Thus, the present findings clearly show that FbpA acts as an important adhesion molecule of L. monocytogenes, especially regarding hepatocytes, without modulating the expression of other virulence factors that have been implicated in the pathogenesis of L. monocytogenes infection.