This study investigated the effects of a 12-week Pilates mat exercise program on blood lipids, aging-related hormones, and follicle-stimulating hormone (FSH) in middle-aged obese women. Twenty women in their 50s with a BMI ≥ 25 kg/m² and no regular physical activity during the previous six months were randomly assigned to a Pilates group (n = 10) or a control group (n = 10). The Pilates program was performed three times per week for 12 weeks, consisting of a warm-up (10 min), main Pilates mat exercise with small equipment (40 min), and cool-down (10 min) at 50–80% HRR. Blood lipids, estradiol (E2), DHEA-S, and FSH were measured before and after the intervention. After 12 weeks, the Pilates group showed decreases in TG and TC, although these changes were not statistically significant. LDL-C significantly decreased, while HDL-C increased without statistical significance. Estradiol significantly increased, whereas DHEA-S showed a non-significant increase. FSH levels increased but did not reach statistical significance. The 12-week Pilates program produced beneficial changes in LDL-C and estradiol and demonstrated generally positive trends in lipid metabolism and aging-related hormones. These findings suggest that Pilates may serve as an effective exercise modality for improving metabolic health and supporting healthy aging in middle-aged obese women.