High-energy expenditure during walking is one of the most important limiting factors for lower limb amputees. Although several physical training methods have been proposed to reduce energy expenditure, little is known about the effects of high-intensity interval training in lower limb amputees. This study aimed to test the effects of a 6-week high-intensity interval training protocol in subjects with lower limb amputation on walking distance, physiological cost of walking, and functional mobility. A quasi-experimental pre and post-study included 13 subjects with lower limb amputation. They performed 12 sessions of a high-intensity interval training protocol with a frequency of 2 days/week for 6 weeks. Walking distance, physiological cost of walking, and functional mobility before and after the training protocol were assessed. Walking distance increased from 204.48 to 229.09 m (P = 0.003), physiological cost of walking decreased from 0.66 to 0.31 beats/m (P = 0.001), and functional mobility improved from 30.38 to 33.61 points (P = 0.001). High-intensity interval training improved walking distance, physiological cost of walking, and functional mobility in subjects with lower limb amputation.