Substrate utilization during exercise is known to differ between children and adults, but whether these differences are related to pubertal status is unclear. The objective of this study was to investigate the effects of pubertal status on endogenous (CHOendo) and orally ingested exogenous (CHOexo) carbohydrate and fat oxidation rates during exercise. Twenty boys at the same chronological age (12 y) were divided into three pubertal groups (pre-pubertal, PP: n = 7; early-pubertal, EP: n = 7; mid- to late-pubertal, M-LP: n = 6) and consumed either a placebo or13C-enriched 6% CHO drink while cycling for 60 min at ~70% of their maximal aerobic power (VO2 max). Another group of 14-year-old boys (pubertal, n = 9) completed all procedures. Substrate utilization was calculated for the final 15 min of exercise using indirect calorimetry and stable isotope methodology. CHOexodecreased fat (p < 0.001) and increased total CHO (p < 0.001) oxidation, irrespective of group. Fat oxidation was higher (p = 0.01) in younger boys than in older boys, but similar (p ≥ 0.33) among PP, EP, and M-LP boys. CHOexocontributed to ~30% of energy expenditure (EE) in PP and EP, but to only 24% in M-LP (p = 0.02), which was identical to the older boys (24%). CHOexooxidation rate as a percentage of EE was inversely related to testosterone levels (r = −0.51, p = 0.005, n = 29). It was concluded that reliance on CHOexoduring exercise is particularly sensitive to pubertal status, with the highest oxidation rates observed in pre- and early-pubertal boys, independent of chronological age.