Using exergaming for exercise training was found to improve physical fitness. Yet, few studies have used the “Xbox Kinect” to examine its effects on physical fitness in healthy middle-aged and older adults. The purpose of this study was to investigate the effect of 10-weeks of Xbox Kinect training on physical fitness in healthy middle-aged and older adults. Forty participants (average 64.00 ± 4.44 years old, eight males and 32 females) were randomized to either intervention (n = 20) or control group (n = 20). The intervention group played Xbox Kinect three times per week, for an average of 50 min per session for 10 weeks. The control group was instructed to maintain their levels of physical activity. All the participants completed assessments of body composition, muscle strength, flexibility, balance and cardiopulmonary endurance at baseline and after 10-week intervention. After 10 weeks of training, the intervention group showed significant improvements in cardiopulmonary endurance and leg muscle strength. Moreover, there were significant differences between the intervention and control group in changes in aerobic fitness and leg muscle strength. The exergame program effectively improved cardiopulmonary endurance and leg muscle strength in healthy middle-aged and older adults. It could be an alternative to conventional exercise.