Having an uncomfortable ride in a vehicle can adversely affect passengers’ travel experience, causing mental and physical fatigue, exacerbating driver fatigue, and increasing the risk of traffic accidents. To determine the level of ride comfort of passenger cars on two-lane mountain highways and the key factors influencing it, in this study, driving tests were performed based on natural driving behavior. Tri-axial acceleration data were collected for various models of small passenger cars, and the peak acceleration values were extracted from the acceleration profiles after they had been filtered using MATLAB filter toolbox. The cumulative frequency curves, characteristic percentiles, and root-mean-square (RMS) values of the tri-axial acceleration were derived. The results revealed that the amplitude of the acceleration, ax, was approximately 50% lower than that of the deceleration, ab. The lateral acceleration, ay, as well as aband ax, varied significantly for the different car models on different roads. The lack of timely maintenance of a few damaged pavements as well as the existence of speed bumps resulted in “considerable discomfort” to the passengers in the form of vertical acceleration, az. The amplitudes of the acceleration along the different axial directions could be ranked in the following order: ay> ab> ax> az; here, ay, ab, and azcontributed the most to passenger discomfort. It was also concluded that the design speed affects ayand absignificantly. Finally, based on the actual experiences of the drivers and passengers, suggestions were made to adjust the thresholds for ax, ay, and az.