Purpose: To improve set-up accuracy in head and neck radiotherapy, a real-time tumor tracking radiotherapy (RTRT) system and a mouthpiece with gold markers has been developed. The reduction in set-up error was estimated in this study. Method and Materials: Four patients with oropharyngeal carcinoma were enrolled in this study. Three 2-mm gold markers were implanted into a mouthpiece, which was designed specifically for each patient. The center of gravity of the three markers and its relationship to the tumor was registered using computed tomography (CT) and 3-D treatment planning system. Translational set-up error was calculated by comparing the actual and planned position of the three markers. Set-up errors by conventional manual immobilization and the RTRT system were compared. Results: In manual set-up, median set-up errors along right-left (RL), cranio-caudal (CC), antero-posterior (AP) directions, and three-dimensional (3D) vector length were 0.5, -0.8, 1.3, and 4.1 mm, respectively (n=87). Systematic set-up errors were 2.8, 3.5, 0.6, and 1.9 mm (n=4). Random set-up errors were 2.6, 3.2, 1.7, and 1.9 mm. In RTRT set-up, translational set-up errors were 0.2, 0.3, -0.3, and 1.6 mm, respectively. Systematic set-up errors were 0.8, 0.8, 0.9, and 0.9 mm (n=4). Random set-up errors were 1.2, 1.5, 1.1, and 1.3 mm (n=92). Statistically significant difference (p<0.001) was seen in random set-up errors of x, y, and 3D vector between the two methods. Conclusion: Set-up errors were improved by using RTRT system with a mouthpiece with three gold markers in head and neck radiotherapy (p<0.001). This system will be useful to improve the accuracy of intensity-modulated radiotherapy (IMRT), which requires meticulous set-up.