Abstract: Objective To analyze the set-up errors of preoperative intensity modulated radiation therapy (IMRT) in locally advanced rectal cancer and its impact factors, and provide important evidence to improve geometric accuracy and radiotherapy precision. Methods Fiftyfive patients with locally advanced rectal cancer treated with preoperative IMRT in Chinese PLA General Hospital from 2014 to 2016 were enrolled. All CBCT images were obtained using on-board imaging system on a Varian Clinac iX linear accelerator. Automatic image registration of the CBCT to the planning CT was performed, and the set-up parameters including left/right (X), superior/inferior (Y), anterior/posterior (Z) directions and rotation (Roll) about X axis were recorded and corrected before radiotherapy implementation. Results All cases had undergone 869 CBCT scans. The average setup errors in the X, Y, Z axes and Roll were(1.50±1.95)mm, (3.05±5.09) mm, (1.42±1.88) mm and (0.8±0.10)°, respectively. The corresponding CTV-to-PTV margins in the direction of X, Y, and Z axes were 5.12 mm, 11.2 mm, 4.87 mm. There were no significant differences between the first 5 and the last 5 set-up and rotation errors (P> 0. 05). Univariate analysis showed that the setup errors of X, Y, Z directions were no significant differences in age, gender, body mass index, tumor distance from anus and tumor which received simultaneous integrated boost (SIB). Conclusion The set-up errors are greater in Y direction than those in X and Z axes during preoperative IMRT of rectal cancer in the supine position and the rotation errors are much smaller and can be omitted.