Objective: To determine the influence of dietary glycemic index on exercise training-induced adaptations in substrate oxidation in obesity. Design and Methods: Twenty older, obese individuals undertook 3 months of fully supervised aerobic exercise and were randomized to low- (LoGIX) or high-glycemic (HiGIX) diets. Changes in indirect calorimetry (VO2 ; VCO2 ) were assessed at rest, during a hyperinsulinemic-euglycemic clamp, and during submaximal exercise (walking: 65% VO2 max, 200 kcal energy expenditure). Intramyocellular lipid (IMCL) was measured by (1) H-magnetic resonance spectroscopy. Results: Weight loss (-8.6 ± 1.1%) and improvements (P <0.05) in VO2 max, glycemic control, fasting lipemia, and metabolic flexibility were similar for both LoGIX and HiGIX groups. During submaximal exercise, energy expenditure was higher following the intervention (P <0.01) in both groups. Respiratory exchange ratio during exercise was unchanged in the LoGIX group but increased in the HiGIX group (P <0.05). However, fat oxidation during exercise expressed in relation to changes in body weight was increased in the LoGIX group (+10.6 ± 3.6%; P <0.05). Fasting IMCL was unchanged, however, extramyocellular lipid was reduced (P <0.05) after LoGIX. Conclusions: A LoGIX/exercise weight-loss intervention increased fat utilization during exercise independent of changes in energy expenditure. This highlights the potential therapeutic value of low-glycemic foods for reversing metabolic defects in obesity.