BACKGROUND: Corn silk is a very important by-product of corn production with medicinal value. Corn silk polysaccharide (CSP) is the main active ingredient. In the present study, ultrasound and spheroidization by anti-solvent were applied to improve the biological activity of CSP. RESULTS: The results showed that ultrasonic degradation improved the α-glucosidase inhibitory activity of CSP by changing its physicochemical characteristics. As the anti-solvent ratio increased, the particle size of the nanoparticles (NPs) from the spheroidization of ultrasonic-degraded corn silk polysaccharide (UCSP) gradually increased, and NP-1 exhibited the highest inhibitory effect of α-glucosidase. Isothermal titration calorimetry (ITC) results indicated that the enhanced activity might be due to more α-glucosidase binding sites with NP-1 compared with no spheroidization. Western blotting results showed that NP-1 could improve the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) uptake in the L6 cells by regulating the phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway and the translocation of glucose transporter 4 (GLUT4). NP-1 also exhibited excellent stability in different environments. CONCLUSION: The study revealed that ultrasonic treatment and spheroidization processing showed potential applications for improving the biological activity of polysaccharides.