Hepatitis B spliced protein (HBSP) is involved in the pathogenicity and/or persistence of hepatitis B virus (HBV). Chronic HBV infection is one of the most important risk factors for the development of hepatocellular carcinoma (HCC). However, whether or not HBSP contributes to the progression of HBV-associated HCC remains unknown. This study reports that overexpression of HBSP in human hepatoma cells increased cell invasion and motility. Conversely, small interfering RNA (siRNA)-mediated knockdown of HBSP expression inhibited migration and invasion. By glutathione S-transferase (GST) pulldown, coimmunoprecipitation, and a mammalian two-hybrid assay, HBSP was found to directly interact with cathepsin B (CTSB). Similar to HBSP knockdown, knocking down CTSB also reduced cell migration and invasion. Furthermore, the HBSP-overexpressing hepatoma cells were shown to have increased expression and activity of matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (uPA), and overexpression of HBSP significantly enhanced tumor-induced vascularization of endothelial cells. In contrast, knockdown of either HBSP or CTSB by siRNA resulted in inhibition of the two proteolytic enzymes and of the in vitro angiogenesis. Expression of HBSP in the hepatoma cells appeared to activate the mitogen-activated protein kinase (MAPK) and Akt signaling pathway, as evidenced by increases in phosphorylation of p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt. Taken together, these findings imply that interaction of HBSP with CTSB may promote hepatoma cell motility and invasion and highlight new molecular mechanisms for HBSP-induced HCC progression that involve the secretion and activation of proteolytic enzymes, increased tumor-induced angiogenesis, and activation of the MAPK/Akt signaling, thereby leading to the aggressiveness of hepatoma cells.