Pyruvate kinase isoenzyme M2 (PKM2) is a key enzyme to regulate aerobic glycolysis in tumor cells and can be used as potential target for cancer therapy. Here, we investigated the effect of knockdown of PKM2 on non-small cell lung cancer (NSCLC) responses to ionizing radiation. Our results showed that PKM2 was greatly up-regulated in radioresistant cell lines and PKM2 knockdown leaded to increased radiosensitivity of radioresistant cell lines, which were associated with higher apoptosis rate and endoplasmic reticulum stress according to western blot analysis. Moreover, significant inhibition in tumor size under regular radiotherapy was found in Balb/c-nude mice bearing radioresistant NSCLC tumors with PKM2 knockdown. Our findings suggested that targeting PKM2 could effectively improve the efficacy of radiotherapy.