5-Fluorouracil-loaded N-succinyl-chitosan nanoparticles (5-FU-Suc-Chi/NP) were prepared by emulsification solvent diffusion. Biodistribution and tumor targeting were evaluated after i.v. administration of 5-Fu-Suc-Chi/NPs in Sarcoma 180-bearing mice. Also, pharmacokinetic profiles were evaluated after intravenous injection of 5-Fu-Suc-Chi/NP via the tail vein to rats. Our experimental results showed the 5-FU-Suc-Chi/NPs could be sustained at a high level in the blood for a very long time, implying its long systemic retention in the circulation. 5-FU-Suc-Chi/NPs were distributed mainly in tumors and liver, with small quantities being found in kidney and spleen. 5-FU-Suc-Chi/NPs accumulated only slightly in the heart and lung, and lowered the toxic effect of 5-FU in the heart and lung. Pharmacokinetic analysis in plasma showed the area under plasma concentration-time curve (AUC), elimination half-life (t_1/2), and residence time (MRT) were increased 2.5-fold, 10.98-fold, and 10.8-fold for 5-FU-Suc-Chi/NP compared with that of free 5-FU, respectively. These results indicate that a long half-life in the circulation and tumor targeting of 5-FU-Suc-Chi/NPs are possible.