Tumor cell invasion is a primary event in the metastatic progression of hepatocellular carcinoma (HCC). Our recent results indicate a concordant elevated expression of osteopontin (OPN) and matrix metalloproteinase-9 (MMP-9) in primary metastatic HCC. This study hypothesizes an MMP-9-directed cleavage of OPN that biologically contributes to HCC metastasis. We found that MMP-9 cleaved OPN into specific fragments in vitro, of which three could be identified by Edman degradation amino-acid sequencing. One of these fragments (OPN-5 kDa, residues 167-210) induced low-metastatic HCC cellular invasion via CD44 receptors, which was effectively blocked by the addition of small peptides within the region of OPN-5 kDa. Increased expression of an OPN splice variant (OPN-c) was associated with clinical metastatic HCC. Overexpression of OPN-c with physiological levels of MMP-9 enhanced cellular invasion and coincided with elevated OPN-5 kDa levels. Our data suggest that an alternative splicing event (OPN-c) promotes extracellular cleavage of OPN by MMP-9, thus releasing a distinct region of OPN (OPN-5 kDa) that is essential for HCC cellular invasion and appears to correlate with metastatic potential. The findings of this study may help to improve advanced-stage HCC prognosis and suggest the utility of small peptides for novel therapies.