BACKGROUND: Starting aerobic exercise with low muscle glycogen content elicits greater fat and less carbohydrate utilization than exercise started with high muscle glycogen content, even when exogenous carbohydrate is ingested during exercise. The mechanisms contributing to greater fat utilization despite ingesting carbohydrate during exercise initiated with low glycogen are not delineated. PURPOSE: Characterize transcriptional regulation of substrate metabolism in response to carbohydrate ingestion during steady-state exercise initiated with low muscle glycogen. METHODS: In a randomized, crossover design, 12 men (mean ± SD, age, 21 ± 4 y; body mass, 83 ± 11 kg; VO2peak, 44 ± 3 mL/kg/min) completed two cycle ergometry glycogen depletion trials, followed by a 24 h period of either high fat (1.5 g/kg carbohydrate, 3.0 g/kg fat) or high carbohydrate (6.0 g/kg carbohydrate, 1.0 g/kg fat) isocaloric refeeding to elicit low (LOW) or adequate (AD) glycogen content the following morning before initiating 80-min of cycle ergometry (64 ± 3% VO2peak) while ingesting 146 g of carbohydrate. Transcriptional regulation of substrate metabolism was assessed using RT-qPCR in vastus lateralis biopsy samples obtained before (PRE) and after (POST) the 80-min exercise bout. RESULTS: PRE glycogen synthase kinase 3α expression was 40% lower (P<0.05; time-by-treatment interaction) in LOW than AD. GLUT4, hexokinase 2, phosphofructokinase, and pyruvate kinase expression were not different between LOW and AD. PRE fatty acid translocase was 40% higher (P<0.05; time-by-treatment interaction) in LOW than AD. Independent of time, fatty acid binding protein, carnitine palmitoyltransferase 1A, and hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA expression were each ~40% higher (P<0.05; time effect) in LOW than AD. In LOW, POST peroxisome proliferator-activated receptor δ was 177% higher (P<0.05 time-by-treatment interaction) than PRE, with no change in AD. CONCLUSION: Initiating aerobic exercise with low muscle glycogen content upregulates the transcriptional control of fat oxidation without modulating intramuscular regulation of glucose metabolism, even when exogenous glucose is ingested during exercise.