Aim: The aim of this study was to prepare a nanosuspension loading a novel insoluble anticancer drug (Q39) by precipitation-high pressure homogenization with poloxamer as stabilizer to improve its solubility, stability and the antitumor activity in vivo.Method: Q39 nanosuspension was prepared by precipitation-high pressure homogenization with poloxamer as stabilizer.The mean particle size and polydispersity index of Q39 nanosuspension were determined by Zetasizer3000HS.The morphology of P188/NS and P85/NS was evaluated by transmission electron microscopy (TEM).The crystalline of drug in different samples was evaluated by X-ray diffraction (XRD).The solubility and stability were evaluated at different condition.The releasing was also evaluated for 6 days.1, 1-dioctadecyl-3, 3, 3, 3-tetramethylindocarbocyanine perchlorate (Dil dye) was used to study the passive targeting of nanosuspension.BABL/c nude mice bearing HepG2 cells were used as tumor models in vivo to evaluate the antitumor activity of Q39 nanosuspension after intravenous administration.And the survival time was calculated to compare the difference.Results: Q39 nanosuspension was successfully prepared by precipitation-high pressure homogenization.The particle size and PI for Poloxamer188 nanosuspension were (304±3) nm, and (0.123±0.005) respectively, and it was (307±5) nm and (0.120±0.007) for Poloxamer85 nanosuspension correspondingly.The crystalline of Q39 during the process didnt change according to the XRD analysis.However, the morphology of two nansouspension was quite different.It was spherical shape for P188 nanosuspension, while elliptoid shape for P85 nanosuspension.The stability of Q39 nanosuspension was improved compared with the solution, and P188 nanosuspension had better stability between P188 nanosuspension and P85 nanosuspension.The solubility of Q39 in P188 nanosuspension was 7.3 times higher than the original solubility, while it was 6 times for P85 nanosuspension.The result of releasing test indicates Q39 nanosuspension had an obvious sustained releasing effect when compared to the solution.The passive targeting of nanosuspension to tumor was demonstrated using Dil as a fluorescence labeling, which may contribute to the enhanced in vivo antitumor activity.The result of antitumor effect of Q39 nanosuspension in vivo also indicates that when the drug was made into nanosuspension, the antitumor activity increased obviously compared to the solution, which may explain the reason why the survival time was prolonged.Conclusion: Q39 nanosuspension was prepared by precipitation-high pressure homogenization method with poloxamer as stabilizer.Compared with the solution, the saturation solubility and stability improved significantly; the crystalline of the drug during the process kept changeless, but the morphology showed a big difference due to the different molecule weight of poloxamer.Q39 nanosuspension had a obvious sustained releasing effect.The antitumor experiment in vivo showed Q39 nanosuspension could inhibit the tumor growth significantly and the survival time was also prolonged when compared to Q39 solution.