Background: Radiotherapy (RT) is a common treatment option for patients with esophagus cancer. Surrounding normal tissue is often receiving scatter RT dose during treatment, which might lead to tissue injuries. Previous studies showed that in addition to directly cause DNA damage, radiation therapy also cause indirect DNA damage, resulting in cell injury or death and further organ dysfunction. Diaphragm usually received certain amount of scatter radiation during RT treatment in patients with esophagus cancer, and the impact of RT on diaphragm contractile function remained to be determined. Purpose: The purpose of this study was to investigate the effect and potential mechanisms of RT on diaphragm contractile function using animal model. Methods: A total of 20 Sprague-Dawley rats were used and randomized into radiation group (RG, n=10) and control group (CG, n=10). Left diaphragms of rats in RG and CG received 5 Gy and 0 Gy (shame) irradiation, respectively, after anesthetized. Before irradiation, baseline pulmonary function (using non-invasive method) was evaluated. Twenty-four hours following irradiation, pulmonary function tests were repeated using both non- and invasive methods. The left diaphragm was removed for contractile function testing. Retention of γH2AX foci after 24 hours of irradiation was analyzed and used as an indicator for DNA damage. Preliminary oxidative stress level was analyzed using CuZnSOD and MnSOD staining methods. SPSS was used for all data analyzes. Generalized Linear Model (GLM) and Generalized Estimating Equation (GEE) model were used to detect differences of variables between and within groups when suitable. Significant α level was set at 0.05. Results: Compared to CG, 24 hours after irradiation, absolute twitch tension (p=0.03), absolute tetanus tension (p=0.03), and specific tetanus tension (p=0.02) was significantly lower in RG. Relative force-frequency curves of RG did not show significant deviation compared to CG. At 24 hours after irradiation, 58% of diaphragm muscle cells remained γH2AX positive, which was significantly higher than in CG (30%, p=0.01). Microscope observation revealed swelling of muscle cells in RG. Staining intensity of CuZnSOD and MnSOD was higher in RG compared to that of CG. Compared to baseline, inspiratory duty cycle (p<0.0001), respiratory rate (p<0.0001), and minute ventilation (RG: p=0.02; CG: p=0.03) were significantly lower after 24 hours of intervention in both groups. Conclusion: Radiation could induce acute (24 hours post irradiation) contractile dysfunction of diaphragm which might be related to RT-induced direct DNA damage and RT-related overwhelmed oxidative stress.