BACKGROUND AND PURPOSE: The aim of this study is to evaluate the effectiveness of a cervical muscle training intervention in decreasing setup errors in patients head and neck cancer (HNC) undergoing radiotherapy (RT). MATERIALS AND METHODS: HNC patients opting for RT at our center. The patients were randomly allocated to either the muscle training group or the control group in a 1:1 ratio. The magnitude of the setup error was measured at the levels of the clivus, C4 and C7 vertebrae respectively. The Van Herk formula was used to determine appropriate planning target volume (PTV) margins. (Trial Registration: ChiCTR2000041009, registration date: 12/16/2020) RESULTS: A total of 221 patients were analyzed, with 109 assigned to the muscle training group and 112 enrolled in the control group. Compared with the control group, the setup errors in the X and Z direction of the clivus and the Z direction of C4 and C7 in the muscle training group were significantly lower (p = 0.031, <
0.001, <
0.001, <
0.001 respectively). The required PTV margins in the Z direction increased from 2.13 mm in the clivus to 3.63 mm in C7 in the muscle training group and from 2.89 mm in the clivus to 4.37 mm in C7 in the control group. Multivariate linear regression analysis demonstrated that the impact of neck muscle training, weight fluctuation, and cervical curvature on the setup error in the Z direction at C7 differed significantly (p = 0.000, 0.001, and 0.008, respectively). CONCLUSION: Neck muscle training can reduce setup errors and PTV margins in the anterior-posterior direction in patients undergoing RT for HNC.