The straddle-type monorail tour transit system is a light overhead steel structure, and the lateral stiffness is generally low. However, the limit of the lateral natural vibration frequency is not clear in the current codes of China, and designers may ignore it. Weak lateral stiffness will lead to a violent vibration during vehicle operation and crowds walking, affecting human comfort and structural safety. Based on a practical project, we tested the acceleration of a monorail vehicle under full load conditions, and its running stability and ride comfort were assessed. Then, the impact of pedestrians on lateral vibration under some working conditions was measured and analyzed. Furthermore, the influence of different structural parameters on the lateral fundamental frequency was investigated. The results showed the following: (i) The vehicle’s running stability and riding comfort was good. However, human comfort was poor due to the weak lateral stiffness of the structure, which was affected by human-induced vibration. (ii) The comprehensive response of the structure increased with the increase in walking frequency, increased with the increase in the number of people working or weight, and the growth speed slowed down. (iii) The structural stiffness was most sensitive to the change in steel column diameter. (iv) The recommended value of the lateral fundamental frequency limit for different spans of the straddle-type monorail tour transit system was put forward. The recommended lower limit of fundamental frequency for a 15 m span is 5.0 Hz, for an 18 m span it is 3.5 Hz, and for a 25 m span it is 2.8 Hz.