To improve the interaction between drivers and the in‐vehicle information system (IVIS), various intelligent agents, such as robot agents, virtual agents, and voice‐only agents, have been integrated into vehicles. However, it is not yet clear which type of in‐vehicle agent is best suited to the driving context. This study aims to investigate the effect of in‐vehicle agent embodiment on drivers' perceived usability and cognitive workload. In a within‐subject simulated driving experiment, 22 participants interacted with three different in‐vehicle agents (smartphone agent, robot agent, and virtual agent). Functional near‐infrared spectroscopy and electrocardiogram (ECG) were used to record prefrontal cortex activation and electrical changes associated with cardiac activity during simulated driving, respectively. The results show that the smartphone agent had the lowest perceived usability scores, oxygenated hemoglobin concentration variation (ΔHbO), and maximum ECG signal variation compared to baseline. There were no statistical differences in cognitive workload, perceived usability scores, brain area activation, and ECG signals between the robot agent and the virtual agent. The research findings demonstrate the positive effects of the anthropomorphic appearance of in‐vehicle agents on perceived usability and contribute to improving the design of in‐vehicle intelligent agents.