Objectives Oral squamous cell carcinoma (OSCC) is able to invade and develop metastasis. Cancer stem cells have been associated with tumor development, progression, and recurrence of OSCC. Tumor cells have high cholesterol rates in their membranes, and cholesterol depletion affects these cells and interferes in carcinogenesis. The objective was to evaluate the effect of cholesterol depletion with methyl-β-cyclodextrin (MβCD) on the expression of stem cell markers CD44 and ESA in OSCC cell line SCC-9. Study Design SCC-9 cells were depleted with MβCD at concentrations 0, 7.5, 10, and 15 mM and evaluated by flow cytometry for CD44 and ESA markers. Results An increase in the CD44-high/ESA-high population was observed after cholesterol depletion at all evaluated concentrations. This increase was accompanied by a decrease in the CD44-low population, indicating that depletion favors the selection of stem cell—like cells. There was no difference in the percentage of CD44-high/ESA-low cells in the treated groups compared to the control group. Conclusions Cholesterol depletion seems to induce a stem cell–like profile with greater proliferative capacity in OSCC cells. Oral squamous cell carcinoma (OSCC) is able to invade and develop metastasis. Cancer stem cells have been associated with tumor development, progression, and recurrence of OSCC. Tumor cells have high cholesterol rates in their membranes, and cholesterol depletion affects these cells and interferes in carcinogenesis. The objective was to evaluate the effect of cholesterol depletion with methyl-β-cyclodextrin (MβCD) on the expression of stem cell markers CD44 and ESA in OSCC cell line SCC-9. SCC-9 cells were depleted with MβCD at concentrations 0, 7.5, 10, and 15 mM and evaluated by flow cytometry for CD44 and ESA markers. An increase in the CD44-high/ESA-high population was observed after cholesterol depletion at all evaluated concentrations. This increase was accompanied by a decrease in the CD44-low population, indicating that depletion favors the selection of stem cell—like cells. There was no difference in the percentage of CD44-high/ESA-low cells in the treated groups compared to the control group. Cholesterol depletion seems to induce a stem cell–like profile with greater proliferative capacity in OSCC cells.