As space agencies prepare for long-duration missions, such as establishing a permanent moon base, maintaining astronauts’ physical and mental health becomes crucial. Exercise is known to counteract the adverse effects of space travel, and virtual reality (VR) has the potential to enhance the psychological well-being of crew members. This study aimed to investigate the impact of bodyweight unloading on speed perception during treadmill walking in a VR environment. Eighteen participants walked on an AlterG treadmill while wearing a VR headset that displayed a moving virtual outdoor environment. The treadmill simulated different bodyweight conditions (100%, 60%, and 20%) to represent Earth’s gravity, intermediate, and lunar gravity, respectively. A staircase method was used to determine participants’ estimated threshold speed for perceiving visual speed equivalence. The results revealed that in all conditions, participants consistently underestimated their walking speed compared to the visual scene speed. No significant differences were found in estimated threshold speeds between the unloading conditions. Individual responses varied, possibly due to biomechanical differences and personal preferences. Participants reported no motion sickness symptoms, likely attributed to the short duration of speed mismatches and the use of high-resolution and high-refresh-rate VR equipment. This study provides insights into the combination of VR and treadmill walking, suggesting the importance of individual customization to enhance user experience and prevent motion sickness.