Summary Legalon SIL ( SIL ) is a chemically hydrophilized version of silibinin, an extract of milk thistle ( Silybum marianum ) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus ( HCV ) in patients leading to viral clearance in combination with ribavirin. To elucidate the incompletely understood mode of action of SIL against HCV , mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA ‐ SCID ‐chimeric mice with humanized livers. Chronically HCV ‐infected mice ( n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albumin ( hA lb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hA lb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV ‐infected hepatocyte decline, δ, was dose‐dependent. Intracellular HCV RNA levels correlated ( r = 0.66, P = 0.01) with serum HCV RNA . Pathway analysis revealed increased anti‐inflammatory and antiproliferative gene expression in human hepatocytes in SIL ‐treated mice. The results suggest that SIL could lead to a continuous second‐phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti‐inflammatory and antiproliferative gene expression in human hepatocytes.