OBJECTIVE: To prepare and evaluate the liver-targeted drug delivery system of Bufalin with higher liver uptake and stronger antitumor activity against hepatocellular carcinoma. METHODS: Bufalin-loaded bovine serum albumin nanoparticle was prepared by desolvation method, to investigate the release performance and to evaluate the pharmacokinetic and tissue distribution. The antitumor activity against hepatocellular carcinoma was evaluated and , respectively. RESULTS: The Bufalin-loaded bovine serum albumin nanoparticle with an average particle size of 125.1 nm exhibited a sustained release behavior . The half-life, blood plasma area under the curve and apparent volume of distribution of Bufalin-loaded bovine serum albumin nanoparticle were significantly higher than that of Bufalin, whereas the clearance rate was lower than Bufalin group. The uptake of liver for Bufalin-loaded bovine serum albumin nanoparticle was 352.045 ± 35.665 ng/g while for Bufalin was 164.465 ± 48.080 ng/g ( 0.01) at 5 min. The uptake of tumor for Bufalin-loaded bovine serum albumin nanoparticle was significantly higher than that of Bufalin both at 5 min (50.169 ± 11.708 ng/g, 93.415±13.828 ng/g, 0.01) and 15 min (43.683 ± 11.499 ng/g, 64.219 ± 17.684 ng/g, 0.05). Bufalin-loaded bovine serum albumin nanoparticle and Bufalin have similar antitumor activity . The tumor inhibition effect of Bufalin-loaded bovine serum albumin nanoparticle was stronger than that of Bufalin alone . CONCLUSION: Bufalin-loaded bovine serum albumin nanoparticle is a promising liver-targeted drug delivery system with higher liver uptake and stronger antitumor activity against hepatocellular carcinoma.