A previous study has reported that knockdown of RING finger protein 2 (RNF2) increases the radiosensitivity of esophageal cancer cells both in vitro and in vivo. However, the effect of RNF2 knockdown on radiosensitivity in squamous cell carcinoma (SqCC) remains unknown. For this, NCI-H226 and SK-MES-1 cells were exposed to X-ray irradiation and then RNF2 levels were determined. RNF2 was knocked-down and stable transfectants were selected. Radiosensitivity, cell proliferation, apoptosis, cell cycle, and gamma-H2AX foci formation were evaluated. Interaction among ataxia telangiectasia mutated protein (ATM), mediator of DNA damage checkpoint 1 (MDC1), and H2AX were examined. Xenograft models were used to explore the effect of RNF2 knockdown on radiosensitivity in vivo. The results showed that RNF2 expression was significantly increased by X-ray irradiation. RNF2 knockdown combined with X-ray irradiation markedly inhibited cell proliferation, caused cell cycle arrest at the G1 phase, and induced cell apoptosis. In addition, RNF2 knockdown enhanced the radiosensitivity of SqCC cells, inhibited irradiation-induced gamma-H2AX foci formation, and impaired the interactions among ATM, MDC1, and H2AX. Furthermore, combination of RNF2 knockdown and X-ray irradiation suppressed tumor growth and promoted tumor cell apoptosis in vivo. RNF2 may be a new therapeutic target to enhance the radiosensitivity of SqCC cells in lung.