Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and poses a severe threat to human health. Ginkgo biloba leaf polysaccharide (GBLP) is a bioactive component, and its sulphated derivative (sulfated GBLP, SGBLP) may exhibit high antitumor activity in certain types of cancers. However, the precise mechanisms of the SGBLP antitumor activity, particularly in HCC, remain unclear. Here, we assessed the effect of SGBLP on HepG2 hepatocellular carcinoma cells. SGBLP was shown to inhibit cellular proliferation and promote apoptosis through the regulation of pro- as well as anti-apoptosis markers, and to induce autophagy by supressing the PI3K/AKT/mTOR pathway. In addition, the autophagy inhibitor 3-melyladenine (3-MA) enhanced the antiproliferative and proapoptotic effects of SGBLP in HepG2 cells. Thus, SGBLP exhibits antitumor activity, and its combination with an autophagy inhibitor may represent a promising anticancer strategy in the treatment of HCC.