The study proposes a new design of the negative stiffness structure using roller-springs (NSS-RS) for the seat suspension to ameliorate the driver’s ride comfort. A mathematical model of the vehicle and seat using NSS-RS is established to simulate the NSS-RS’s vibration characteristics under various working conditions. Then, the design parameters of NSS-RS are optimized based on the genetic algorithm to improve the driver’s ride comfort. Two indexes of the root-mean-square of the seat displacement (zws) and seat acceleration (aws) are chosen as the objective function. The results show that the seat suspension using NSS-RS greatly improves the driver’s ride comfort compared to the traditional seat suspension under all operating conditions of the vehicle. Particularly, under a random excitation, the values of zws and aws using NSS-RS are significantly reduced by 53.6% and 82.3% in comparison with the traditional seat suspension. Consequently, the seat suspension using the NSS-RS should be applied to further enhance the driver’s ride comfort.