In view of the benefits and a promising prospect of the emerging connected vehicles (CV), more and more vehicles will be equipped with the in-vehicle human-machine interface (HMI). This study aims to assess the comprehensive impacts of HMIs on individual driving performance, traffic safety, and eco-driving behaviour based on a driving simulator. In order to explore how a driver responds to warnings when approaching a tunnel, this study divided the experimental road into five zones according to warning positions. A comparative analysis was conducted to examine the changes in driving comfort, speeding behaviour, the standard deviation of speed, time headway (THW), fuel consumption, and emissions. Results showed that the HMI could be beneficial to speed adjustment before entering a tunnel. Meanwhile, HMI may affect the THW of vehicles. Drivers were usually more cautious when the HMI was on. However, no significant effects of the HMI for driving comfort and eco-driving behaviour can be observed. It is expected that more efficient technology and different warning strategies can be developed to enhance eco-safe driving for different zones because they have different effects. The findings of this study are also helpful for active safety management and for evaluating the effectiveness of CV systems. 1 INTRODUCTION Traffic systems are composed of human, vehicle, road, infrastructure, and environment, in which drivers are the main part. Previous research has consistently shown that inappropriate human manoeuvre is responsible for about more than 80% of all traffic crashes and traffic congestions [1-3]. Human factors are an important topic on the way of seeking for traffic safety and efficiency. The development of information and communication technologies has provided opportunities to adjust driving behaviour in improving traffic safety and capacity and in reducing fuel consumption and emissions [4-7]. Connected vehicle (CV) technologies, in which vehicles communicate with other vehicles, roadway infrastructure, and traffic management entity in real time, can give abundant and accurate information to drivers. These information help drivers make more preparations for the upcoming traffic emergency events or the changing environments of driving situations. Road tunnels are usually designed to reduce travel time and protect natural resources and the environment. The number of road tunnels is increasing rapidly due to the above advantages This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.