Hepatocellular carcinoma (HCC) is a serious cancer with high mortality worldwide. Gemcitabine (GEM) is easily degraded in the circulation and has no tumor-targeted effect. In our previous research, an amphiphilic GEM derivative, cyclic phosphoryl N-dodecanoyl gemcitabine (CPDG) was prepared based on the techniques of HepDirect prodrug and self-assembled drug delivery systems (SADDS), which self-assembled into the stable nanoassemblies in water. In this study, the long-circulating nanoassemblies of CPDG/CHS-PEG1500 (9:1, mol/mol) were prepared for HCC treatment. In vitro and in vivo studies of the long-circulating CPDG nanoassemblies were explored. The degradation rates of CPDG depended on the media. CPDG showed much faster degradation in the acidic environment (pH 2.0) than the weak acidic and neutral media (pH 5.0, pH 7.4). However, the degradation half-life (t_1/2) of CPDG was about 43h in the mouse plasma, longer than the t_1/2 at pH 2.0. Therefore, the long-circulating CPDG nanoassemblies could keep stable before reaching the targets in vivo. In the biodistribution study, the long-circulating CPDG nanoassemblies were bolus intravenously (i.v.) injected into the hepatocellular tumor-bearing mice. The distribution of CPDG in the tumors was much higher than that in the blood, indicating the tumor targeting of the long-circulating nanoassemblies. In the pharmacodynamic study, the long-circulating CPDG nanoassemblies were i.v. injected into the tumor-bearing mice with doses of (37.5, 75μmol/kg) compared with GEM (150μmol/kg). The mice were injected once every 3days for totally 3 times. The long-circulating nanoassemblies nearly always showed the higher anti-cancer effects than GEM. The tumor inhibitory rates of GEM, the long circulating CPDG nanoassemblies (37.5, 75μmol/kg) were 49.54, 42.97, 65.10%, respectively. Therefore, the long-circulating CPDG nanoassemblies had the much higher anti-cancer effect than GEM. The long-circulating CPDG nanoassemblies are promising nanomedicines to treat HCC. The combination design of tumor-targeted nanoassemblies based on HepDirect prodrug technique and SADDS theory is an effective method to modify the pharmacologically active nucleosides to treat some liver diseases.