Context Custard apple (Annona squamosa Linn.) is an edible tropical fruit, and its seeds was used to treat "malignant sore" (cancer) and other usage as insecticide.A comparison of extraction processes, chemical composition analysis and antitumor activity of A.squamosa seeds oil (ASO) were investigated.Materials and methods The optimal extraction parameters of ASO were established by comparing percolation, soxhlet, ultrasonic and SFE-CO2 extraction methods.The chemical composition of fatty acid and content of total ACGs of ASO was investigated by GC-MS and colorimetric assay, and anti-tumor activity of ASO was tested using H22 xenografts bearing mice.Results The optimal extraction parameters of ASO were obtained as follows: using soxhlet extraction method with extraction solvent of petroleum ether, temperature of 80 ℃, and extraction time of 90 min.Under these conditions, the yield of ASO was 22.65%.GC-MS analysis results showed that the main chemical compositions of fatty acid of ASO were palmitic acid (9.92%), linoleic acid (20.49%), oleic acid (56.50%) and stearic acid (9.14%).The total annonaceous acetogenins (ACGs) contents in ASO were 41.00 mg/g.ASO inhibited the growth of H22 tumor cells in mice with a maximum inhibitory rate of 53.54% by oral administration.Furthermore, it was found that ASO exerted an antitumor effect via decreasing interleukin-6 (IL-6), janus kinase (JAK) and phosphorylated signal transducers and activators of transcription (pSTAT3) expression.Discussion and Conclusion The results demonstrated that ASO suppressed the H22 solid tumor development may due to its main chemical constituents unsaturated fatty acid and ACGs via IL-6/JAK/STAT3 pathway.ASO may be a potential candidate for the treatment of cancer.