This study examined substrate use during exercise in early-pubertal (EP), mid-pubertal (MP), late-pubertal (LP), and young-adult (YA) males. Fuel use was calculated using the RER and VO2 response during cycling exercise at 30 to 70% of VO2speak. Significant group by intensity interactions were found for lactate, RER, percent CHO, and fat use, in addition to fat and CHO oxidation rates, which suggest a maturation effect on substrate use during exercise. While significance was not achieved at all intensities, post hoc analyses revealed greater fat use, lower CHO use, and lower lactate concentrations in EP and MP compared to LP or YA. No differences were noted between EP and MP or LP and YA at any intensity, suggesting the development of an adult-like metabolic profile occurs between mid- to late-puberty and is complete by the end of puberty.