The present study investigated the production, in vivo biodistribution and hepatoprotective of two formulated silybin nanosuspensions with different particle size. The physicochemical properties of the two formulated silybin nanosuspensions were investigated by TEM, AFM and SEM. A kinetic study was conducted to evaluate the influence of particle size on the in vivo tissue distribution following intravenous administration in the mice. The in vivo hepatoprotective studies were conducted on beagle dogs with optimized setting. A clear physicochemical difference was observed among the silybin solution, larger particles and the small particles. The formulation of larger particle size was preferentially targeted at liver and spleen. The silybin nanosuspensions, administrated either intravenously or orally, presented significant (P < or = 0.05) hepatoprotective effect by reducing the serum marker enzymes such as AST, ALT, ALP, TBIL and GGT. Histopathological study further confirmed the hepatoprotective activity of the two silybin nanosuspensions formulations when compared with the CCl4 treated control group. These results indicate that the nanosuspensions approaches could be used to improve the drug target delivery and therapeutic efficacy of the silybin.