The expression of αV integrins by neoplastic cells contributes to the promotion of local invasion and metastasis. The most characteristic extracellular ligands of αV integrins are vitronectin and fibronectin. Hepatocytes are the main source of vitronectin, and the capacity to synthesize and secrete vitronectin is usually retained in hepatocellular carcinoma. The aim of this study was to explore the expression, regulation, and functional role of αV integrins in hepatocellular carcinoma. We first analyzed the expression of αV integrins and their ligands fibronectin and vitronectin in 80 cases of hepatocellular carcinoma. αV integrin chain was detected in 44 cases and vitronectin in 50. Twenty-four of the 44 αV-positive tumors contained large amounts of vitronectin. These cases presented more frequently with adverse histoprognostic factors, including infiltrative growth pattern (62.5%), lack of capsule (71%), presence of capsular invasion (57%), and satellite nodules (50%). We then used HepG2 and Hep3B cell lines as in vitro models to study αV integrin regulation and function. HepG2 and Hep3B cells expressed αV integrin chain and used αVβ1 and αVβ5 for adhesion and migration on vitronectin. Tumor necrosis factor (TNF) α and transforming growth factor (TGF) β significantly increased the expression levels of αV integrins and stimulated the adhesion and migration of both HepG2 and Hep3B cell lines on vitronectin. The effects of growth factors on cell adhesion and migration were reproduced by incubation with conditioned medium from rat liver myofibroblasts. In conclusion, our results support the existence of an αV integrin/vitronectin connection in hepatocellular carcinoma and suggest that this connection may be an adverse prognostic factor.