Among the differentially expressed proteins, S100 calcium binding protein A6 (S100A6), glutamine gamma‐glutamyltransferase 2 (TGM2), glycogen phosphorylase, brain form (PYGB), and Thymosin Beta 10 (TMSB10) were selected for further validation studies as they were found to be over‐expressed in the accumulated radio‐resistant ESCC cells and radio‐resistant cells. The gene discussed is PYGB; the disease is esophageal squamous cell carcinoma.