Considering anti-aging and preventive effects of Exercise and Nutritional interventions, the present study was carried out to demonstrate combined effects of 12 weeks of Pilates exercise training and daily turmeric supplementation on serum Klotho level and health related quality of life in overweight middle-aged women. Forty-eight post-menopausal overweight women (mean age: 50. 3 ± 3. 8 years and BMI: 25-30 kg/m2) were selected and randomly divided into four groups of Pilates exercise, turmeric supplementation, combination of both and control. Each Pilates training session (12 weeks, three sessions per week) consisted of 45 minutes of exercise with an intensity of 11-13 Borg RPE scale. The supplementation contract included daily receiving 2, 100 mg of turmeric powder. Blood samples were obtained 48 hours before and after the intervention and serum Klotho levels were measured. HRQL was assessed by using short form 12 (SF-12) questionnaire, completed before and after intervention. Data analysis was performed by t-test and ANOVA (p≤ 0. 05). Results showed that 12 weeks of Pilates training with and without turmeric supplementation significantly increased Klotho levels and improved HRQL, physical health, mental health and social function, but turmeric supplementation alone, did not have significant effect on these factors (P> 0. 05). Collectively, these findings highlight the beneficial effects of Pilates training on Klotho level and HRQL in middle-aged women.