Colon cancer is one of the leading causes of illness and death in the world, with almost 2 million new cases per year, and the third most frequently diagnosed form of cancer. The aim of this study was to demonstrate the effect of 18β-glycyrrhetinic acid (18β-GA) and stearyl glycyrrhetinate (SG) on HCT116 colon cancer cells. The bioactive pentacyclic triterpenoid, 18β-glycyrrhetinic acid and its stearyl ester SG, showed a decrease in the cell viability of HCT116 cells (colon cancer) depending on the applied dose, using the cell viability analysis (XTT test). 18β-GA achieved an IC50 value of 245,08 µM while SG achieved a fivefold lower IC50 value of 35,28 µM. Considering the better effect in reducing the viability of SG tumor cells compared to 18β-GA, in a lower dose, the mechanisms by which SG achieves its antitumor effect have been demonstrated. Western blot analysis showed the effect of SG on the change in the expression of the protein of interest. SG, dose-dependently (10, 15, 20 and 25 µM) increased the expression of the cell cycle inhibitor, p21. The expression of p-AMPKα Thr172 (AMPKα activation), depending on the dose of SG, increased, indicating a decrease in the amount of ATP in the cells, i.e. an increase in the AMP/ATP ratio. Spectrophotometric analysis of glucose consumption by measurement in the cell medium showed that treatment with SG reduces glucose consumption in a dose-dependent manner. Accordingly, SG increased the expression of the autophagy protein, LC3B II. Analysis of the expression of PARP, a marker of apoptosis in case of its cleavage, showed that the cells did not activate apoptosis, but autophagy. Co-treatment with 3-methyladenine (3-MA), an autophagy inhibitor, showed a protective effect, suggesting activation of cytotoxic autophagy in SG treatment. The results of the current research provide insight into the mechanism of action and the antitumor potential of SG in the treatment of colon tumors through the activation of cytotoxic autophagy.