Introduction: There are fewer mitochondria and a reduced oxidative capacity in skeletal muscle in obesity. Moderate-intensity physical activity combined with weight-loss increases oxidative enzyme activity in obese, sedentary adults, however, this adaptation occurs without a significant increase in mtDNA which is unlike the classic pattern of mitochondrial biogenesis induced by vigorous activity. Objective: To examine the hypothesis that the mitochondrial adaptation to moderate-intensity exercise and weight loss in obesity induces increased mitochondrial cristae despite a lack of mtDNA proliferation. Methods: Content of cardiolipin and mtDNA and enzymatic activities of the electron transport chain (ETC) and Krebs cycle were measured in biopsy samples of vastus lateralis obtained from sedentary obese men and women before and following a 4 month walking intervention combined with weight-loss. Results: Cardiolipin increased by 60%, from 47±4 to 74±8 µg/mU CK (p<0.01), but skeletal muscle mtDNA content did not change significantly (1901±363 to 2169 ± 317). Enzyme activity of the ETC increased (p<0.01); that for rotenone-sensitive NADH-oxidase (96±1%) increased more than for ubiquinol-oxidase (48±6%). Activities for citrate synthase (CS) and succinate dehydrogenase (SDH) increased by 29±9% and 40±6%, respectively. Conclusion: Moderate-intensity physical activity combined with weight-loss induces skeletal muscle mitochondrial biogenesis in previously sedentary obese men and women, but this response occurs without mtDNA proliferation and may be characterized by an increase in mitochondrial cristae.