Abstract: Annonaceous acetogenins (ACGs) are effective part extracted and separated from Annona squamosa seeds, they have good antitumor activity against a variety of tumor cells. However, the solubility of ACGs is poor with serious toxic and side effects, which greatly limits their application in clinical practice. In this study poloxamer 188 (P188) was selected as a drug carrier or a stabilizer to prepare ACGs nanosuspensions (ACGs-NSps) using anti-solvent precipitation. The nanosuspensions were examined via dynamic light scattering (DLS) method to examine size of the nanosuspensions. Transmission electron microscopy was used to observe their morphology. HPLC assay was used to measure their drug loading content and the in vitro drug release. The stability of ACGs-NSps at room temperature, in various physiological media and plasma, and the hemolytic test and lyophilization were all investigated. MTT assay was performed to study the cytotoxocity of ACGs-NSps against four tumor cell lines. 4T1 bearing tumor model was used to assess their in vivo antitumor therapeutic efficacy. The obtained ACGs-NSps were spherical, the average particle size was 169.4±1.25 nm, the polydispersity index (PDI) value was 0.130±0.020, the zeta potential was -19.8 mV and the drug loading content was 48.18%. ACGs-NSps were stable at room temperature for at least 15 days. They could be lyophilized in the presence of 0.5% glucose and 2.0% P188. ACGs-NSps showed sustained in vitro drug release, and the cumulative drug release reached 80.82% within 144 hours. ACGs-NSps maintained their particle size in various physiological media, and plasma with no hemolysis and then met demands of both oral and intravenous administration. In contrast to free ACGs, ACGs-NSps displayed significantly higher cytotoxicity against 4T1 (IC₅₀, 0.892±0.124 μg·mL^-1 vs 2.495±0.108 μg·mL^-1, P < 0.05), HeLa (IC₅₀, 0.747±0.051 μg·mL^-1 vs 2.204±0.064 μg·mL^-1, P < 0.01), HepG2 (IC₅₀, 2.265±0.081 μg·mL^-1 vs 4.159±0.071 μg·mL^-1, P < 0.01), and MCF-7 (IC₅₀, 0.473±0.024 μg·mL^-1 vs 1.196±0.022 μg·mL^-1, P < 0.05). The in vivo study demonstrated that the daily oral administration of ACGs-NSps (3 mg·kg^-1) resulted in higher tumor inhibition rate compared to ACGs/oil solution (67.23% vs 53.11%), comparable to the intravenous injection of 0.5 mg·kg^-1 ACGs-NSps every other day (70.34%). Nanosuspensions effectively solved the problem of ACGs insolubility and difficulty in drug delivery. Using P188, a pharmaceutic adjuvant approved by FDA for iv injection, the resultant ACGs-NSps appear promising as an anti-tumor drug that can be used in clinic.