Introduction. Esophagus squamous cell carcinoma (ESCC) has a poor prognosis, a high rate of metastasis, and rapid clinical progression. One hypothesis is that therapeutic failure is due to the presence of cancer stem cells (CSC). Previous studies showed the anticancer effect of cerium oxide nanoparticles (CNP) in different cancer cells. In this study, we aim to evaluate the effect of cerium oxide nanoparticles on cell antioxidants, toxicity, as well as cell oxidant level in esophageal cancer (YM1) and cancer stem cell‐like (CSC‐LC) cell lines. Method. YM1 and CSC‐LC spheres were treated with CNP at different concentrations. The cell viability was assessed by using the MTT test. Antioxidant levels (SOD (superoxide dismutase, CAT (catalase), thiol, and TAC (total antioxidant capacity)), antioxidant genes expression (SOD and CAT), ROS (reactive oxygen species), and MDA (malondialdehyde) levels were assessed in both cell lines. Results. CSC‐LC had significantly elevated SOX4 and OCT4 pluripotent genes. The ROS and MDA levels were significantly reduced in both YM1 and CSC‐LC spheres after treatment with CNP. Also, the antioxidant levels and expressions were elevated significantly in both cell lines after CNP treatment. Conclusion. These results suggest the potential anticancer effect of CNP by elevating antioxidant levels and expressions, and reducing oxidant levels.