Recently osteopontin (OPN), a protein of the extracellular matrix, has generated in hepatocellular carcinoma (HCC) a significant interest as a prognostic factor. Aim of this study was to confirm, in liver tissues of subjects with HCV-positive HCC undergoing liver transplantation (RL, n = 10) and of donors (DL, n = 14), the increase of OPN plasma and tissue concentration, the OPN splicing isoforms expression profiling together with those of thrombin, and to evaluate a possible association between OPN measurements. Their association with Notch-1, IV-Collagen-7s domain, IL-6 and TNF-α were also evaluated. Real-Time PCR experiments and immunometric assay were performed. mRNA expression resulted higher in RL than in DL for all analyzed genes and several correlations were found between them. The more relevant association were between OPN-a and OPN-b (p < 0.0001), between thrombin and OPN-a (p = 0.007), between 7s-collagen and OPN isoforms (p < 0.05) and between Notch-1 with OPN-c (p = 0.004). Both OPN plasma and liver tissue extract concentrations were assessed confirming the trend observed at the mRNA level. An important association was found between OPN plasma and protein (p < 0.0001, r = 0.96) even splitting patients in DL (p < 0.0001, r = 0.93) and RL (p < 0.0001, r = 0.96). A reduction of OPN plasma levels was found at 6 months after transplantation. Considering MELD score as liver disease severity, the mRNA expression of our markers as well as of OPN plasma and tissue concentrations resulted increased as a function of clinical severity. Our results might be considered a useful starting point to validate OPN as a prognostic and diagnostic marker of HCC.