This study investigated the middle cerebral artery blood velocity (MCAv) response to constant work-rate moderate-intensity cycling exercise in 21 children (9.3 ± 0.8 yr), 17 adolescents (12.3 ± 0.4 yr), and 20 young adults (23.6 ± 2.4 yr). Participants completed an incremental ramp test to exhaustion on a cycle ergometer to determine maximal oxygen uptake and gas exchange threshold (GET) before completing three 6-min transitions at a moderate intensity (90% GET) on separate visits. On each visit, bilateral MCAv was measured by transcranial Doppler ultrasonography and breath-by-breath end-tidal carbon dioxide ([Formula: see text]) via a metabolic cart. Data were ensemble-averaged for each participant and analyzed using a monoexponential model. Absolute MCAv was significantly higher throughout exercise in children and adolescents compared with adults (P < 0.001). Children had a significantly lower relative increase in MCAv from baseline (∼12%) compared with adolescents (∼20%) and adults (∼18%, P < 0.040). All adolescents and adults had a monoexponential rise in MCAv and [Formula: see text], but this was observed in only eight children. Children and adolescents had a significantly faster MCAv time constant (τ, 12 ± 6 and 14 ± 8 s, respectively) compared with adults (27 ± 9 s, P < 0.001). MCAv τ was positively associated with faster [Formula: see text] τ in adolescents (r = 0.70, P = 0.002) but not in children (r = -0.20, P = 0.640). Time- and amplitude-based response parameters of MCAv kinetics were significantly associated with [Formula: see text] kinetics in adults (r = 0.50-0.74, P ≤ 0.025), but not in children (r = -0.19 to -0.48, P > 0.227). These findings suggest that the transition from childhood to adulthood impacts the MCAv response to exercise and the relationships between [Formula: see text] and MCAv kinetics during exercise.NEW & NOTEWORTHY This is the first study to find that children have smaller increases in Δ%MCAv (∼12%) during moderate-intensity exercise compared with adolescents and adults (∼18%-20%). Furthermore, MCAv kinetics were significantly faster in children and adolescents, compared with adults. MCAv kinetic responses were significantly and positively associated with [Formula: see text] kinetics in adults, but not in children. These novel data also suggest that the regulatory role of [Formula: see text] on MCAv during exercise begins to strengthen during adolescence.