The serine protease urokinase-type plasminogen activator (u-PA) is overexpressed in hepatocellular carcinoma (HCC) and its expression level is inversely correlated with the patients' survival. The purpose of this study was to examine the effects of vector-based RNA interference (RNAi) of u-PA on the growth of human HCC xenografts in nude mice in order to investigate the role of u-PA in human HCC. Our results showed that the subcutaneous injection of small interfering RNAs (siRNA) u-PA SKHep1C3 stable transfected cells (pS siRNA u-PA) led to a growth delay in xenograft development, compared to those generated from empty vector; the molecular characterization of nodules (carried out by PCR, RT-PCR and immunohistochemical analysis) revealed the presence of plasmid DNA, the u-PA gene expression knockdown, at both mRNA and protein levels, giving evidence of a long-term and target-specific inhibition by vector-based RNAi 11 weeks after cell inoculation. We further studied the effects of u-PA down modulation on extracellular matrix (ECM) proteins evaluating the expression and organization of fibronectin (FN; one of the main ECM proteins). Immunohistochemical and immunofluorescence analysis of FN revealed FN fibrils in pS siRNA u-PA xenografts and in pS siRNA u-PA cells, thus identifying the FN fibril organization as a downstream effect of u-PA knockdown in this system.