Objective This study investigated the effects of different approach directions, movement speeds, and trajectories of a co-robot’s end-effector on workers’ mental stress during handover tasks. Background Human–robot collaboration (HRC) is gaining attention in industry and academia. Understanding robot-related factors causing mental stress is crucial for designing collaborative tasks that minimize workers’ stress. Methods Mental stress in HRC tasks was measured subjectively through self-reports and objectively through galvanic skin response (GSR) and electromyography (EMG). Robot-related factors including approach direction, movement speed, and trajectory were analyzed. Results Movement speed and approach direction had significant effects on subjective ratings, EMG, and GSR. High-speed and approaching from one side consistently resulted in higher fear, lower comfort, and predictability, as well as increased EMG and GSR signals, indicating higher mental stress. Movement trajectory affected GSR, with the sudden stop condition eliciting a stronger response compared to the constrained trajectory. Interaction effects between speed and approach direction were observed for “surprise” and “predictability” subjective ratings. At high speed, approach direction did not significantly differ, but at low speeds, approaching from the side was found to be more surprising and unpredictable compared to approaching from the front. Conclusion The mental stress of workers during HRC is lower when the robot’s end effector (1) approaches a worker within the worker’s field of view, (2) approaches at a lower speed, or (3) follows a constrained trajectory. Application The outcome of this study can serve as a guide to design HRC tasks with a low level of workers’ mental stress.