Empirical evidence suggests that three-dimensional (3D) images of nature promote physiological relaxation in humans by providing more realistic effects compared with two-dimensional (2D) images. However, no studies have evaluated the physiological relaxation effects of nature-derived 3D images on prefrontal cortex and autonomic nerve activity. The present study aimed to clarify the physiological relaxation effects of visual stimulation by 3D flower images on prefrontal cortex and autonomic nerve activity. Nineteen male university students (22.2 +/- 0.6 years) were presented with 3D and 2D images of the water lily for 90 s. Prefrontal cortex activity was measured using near-infrared spectroscopy, while autonomic nerve activity was measured using heart rate variability (HRV). Psychological effects were determined using a modified semantic differential method (SD). Compared with visual stimulation by 2D images, that by 3D images resulted in a significant decrease in oxyhemoglobin concentration in the right prefrontal cortex, lower sympathetic activity as calculated by the ratio of the low-frequency to high-frequency HRV component, and a significantly greater realistic feeling as evidenced by higher SD ratings. In conclusion, visual stimulation by realistic 3D floral images promotes physiological relaxation more effectively than the corresponding 2D image.