Driver fatigue is a significant contributing factor to numerous traffic crashes, which brings great socioeconomic concerns to policy makers, the general public as well as transportation professionals. There is an emerging consensus that the monotonous road environments are the major exogenous factor causing driver fatigue. This study aims to reveal the relationship between the road environment and driver fatigue. Furthermore, the optimal stimulation interval of the road environment to prevent driver fatigue is proposed. A driving simulated experiment study is conducted to evaluate the impact of the monotonous road environment on driver fatigue. The experimental scenario of the road environment is designed based on the real-life environment. The physiological indicator of heart rate (HR) is used to measure driver fatigue. The MPEG (Moving Picture Expert Group) video compression technique is applied to assess the monotony of the road environment. Furthermore, the changing pattern of driver fatigue under different levels of monotony of the road environment, which can be reflected by different stimulation densities, is exhibited. In this study, the relationship between driver fatigue and stimulation density is established. The results of the study are consistent with the Hancock and Warm U model. The results of this study suggest that the optimal stimulation interval in the road environment should be no more than 8 minutes. The findings are further discussed with reference to the design of the road environment in order to mitigate driver fatigue. The potential application of this research is to develop an evaluation system of the road environment based on driver fatigue.