Objective: The aim of our study was to examine the effects of a 6-month school-based high-intensity interval training intervention (Burn 2 Learn [B2L]) on prospectively-defined regional brain volumes in adolescents using magnetic resonance imaging (MRI). Methods: Participants were older adolescents [N=56, 61% female, 16.1±0.4 years] from four schools (10 classes) participating in a larger cluster randomised controlled trial. Participants were randomised to B2L (five classes, N=30) or a control group (five classes, N=26) and underwent MRI at baseline and 6-month follow-up. Changes in regional brain volumes were assessed using linear mixed models adjusted for sex, weight status, intracranial volume, and class level clustering. Results: We observed group-by-time increases in total (+105.5 mm 3, 95% confidence interval [CI] 25.2 to 185.8, d=0.79) and left (+64.7 mm 3, 95% CI 13.8 to 115.7, d=0.76) hippocampal volume, as well as left medial superior frontal gyrus volume (+144.8 mm 3, 95% CI 17.1 to 272.5, d=0.68) in favour of the B2L intervention. The B2L intervention decreased total (-30.6 mm 3, 95% CI -50.9 to -10.2, d=-0.90), left (-15.2 mm 3, 95% CI -28.9 to -1.5, d=-0.67) and right (-18.6 mm 3, 95% CI -30.8 to -6.4, d=-0.92) nucleus accumbens volume, as well as total (-78.3 mm 3, 95% CI -146.8 to -9.9, d=-0.69) and right (-58.7 mm 3, 95% CI -99.7 to -17.6, d=0.86) putamen volume. Conclusion: This is the first experimental study to show that physical activity impacts regional brain volumes in adolescents. Larger studies are needed to replicate our findings and confirm the effects of exercise training on brain health.