The purpose of the present study was to investigate the effect of glycemic index (GI) and fructose content in lunch on substrate utilization during subsequent brisk walking. Ten healthy young males completed 3 main trials in a counterbalanced crossover design. They completed 60 min of brisk walking at approximately 50% maximal oxygen consumption after consuming a standard breakfast and 1 of 3 lunch meals, i.e., a low GI meal without fructose (LGI), a low GI meal that included fructose beverage (LGIF), or a high GI meal (HGI). The 3 lunch meals were isocaloric and provided 1.0 g·kg⁻¹ carbohydrate. Substrate utilization was measured using indirect respiratory calorimetry method. Blood samples were collected at certain time points. During the 2-h postprandial period after lunch, the incremental area under the blood response curve values of glucose and insulin were higher (p < 0.05) in the HGI trial than those in the LGI and LGIF trials (HGI vs. LGI and LGIF: glucose, 223.5 ± 24.4 vs. 92.5 ± 10.4 and 128.0 ± 17.7 mmol·min·L⁻¹; insulin, 3603 ± 593 vs. 1425 ± 289 and 1888 ± 114 mU·min·L⁻¹). During brisk walking, decreased carbohydrate oxidation was observed (p < 0.05) in the LGI trial than in the LGIF and HGI trials (LGI vs. LGIF and HGI: 60.8 ± 4.0 vs. 68.1 ± 6.0 and 74.4 ± 4.7 g). No difference was found in fat oxidation among the 3 trials (LGI vs. LGIF vs. HGI: 21.6 ± 2.3 vs. 19.2 ± 2.3 vs. 16.4 ± 2.2 g). It appeared that fructose content was an important influencing factor when considering the effect of different GI lunch meals on substrate utilization during subsequent moderate intensity exercise.