Raised pavement markers (RPMs) are among the common safety features of roads, playing an important role in preventing and reducing traffic crashes. RPMs are regarded as an effective measure for reducing the high crash rate and mortality in freeway tunnels in China. In this study, a driving simulator experiment was conducted to investigate the safety of RPMs in a freeway tunnel. Two different RPM layouts were designed and compared to a control with no RPMs, and 32 drivers participated in the driving simulator experiments. The speed, relative speed difference, lateral position, accelerator power, acceleration, and pupil area were used as indicators of the response characteristics of drivers to RPMs, and the interaction of tunnel length, tunnel zone, and RPM alternatives was discussed. The results indicate that a significant interaction effect exists between tunnel length, tunnel zone, and RPM alternatives. RPMs could help reduce driver anxiety, boredom, and fatigue caused by the dark and monotonous tunnel driving environment, and improve driver alertness and consciousness of speed. Also, the driving risk increases with increasing tunnel length (1800 m to 3500 m).