OBJECTIVE: To explore the effect of the composition ratio on substitution of sulfate group in sulfated exopolysaccharide (EPS) from and how sulfate modification affects the anti-tumor activity of EPS. METHODS: We used a chlorosulfonic acid-pyridine method to modify EPS and analyzed the effect of esterification ratio on the degree of sulfate substitution using barium chloride turbidimetry. The sulfate groups binding with EPS were analyzed with infrared spectrum analysis. CCK-8 assay was used to evaluate the inhibitory effect of EPS sulfate (SEPS) on the proliferation of human colon cancer HCT 116 cells, and annexin V-FITC/PI double staining was used to assess the pro-apoptotic effect of SEPS in the cells. RESULTS: The esterifying agent and EPS at the composition ratios of 1:1 and 2:1 resulted in sulfate substitution of 0.98% (SEPS-1) and 1.18% (SEPS-2), respectively, and the substitution was improved by increasing the ratio of the esterifying agent ( < 0.05). Infrared spectrum analysis showed that the S=O stretching vibration absorption peak of -OSO appeared near 1249 cm, indicating that the sulfate group combined with EPS to form sulfate. CCK-8 assay showed that SEPS-1 produced stronger inhibitory effects on the proliferation of HCT 116 cells than EPS within the concentration range of 0.02-0.10 mg/L ( < 0.05). At the concentrations of 0.04-0.08 mg/L, SEPS-2 showed a lower anti-tumor activity than SEPS-1 ( < 0.05). SEPS-1 also showed stronger pro-apoptotic effect than EPS, and as its concentration increased, SEPS-1 dose-dependently increased the ratio of early apoptotic cells and necrotic cells; the cells treated with 0.06, 0.08 and 0.10 mg/mL SEPS-1 showed early apoptotic rates of 6.38%, 11.8% and 12.5%, and late apoptotic and necrotic rates of 5.26%, 8.04% and 6.80%, respectively. CONCLUSIONS The composition ratio of the esterifying agent has a direct impact on the degree of substitution of EPS, which can be improved by increasing the ratio of the esterifying agent. Sulfate modification of EPS can enhance its antitumor activity, which, however, is not directly related with the degree of substitution.