Background: This study aimed to evaluate the role of biological age in the relationship between preintervention fat mass and cardiorespiratory fitness effects (CRF) after High-Intensity Interval Training (HIIT) intervention in adolescents. Methods: A total of 141 boys and girls (16 years) were examined as control (CG) and experimental (EG) groups that conducted a 10-week HIIT implemented in physical education. Measurements: body height, body weight, fat mass index (FMI), CRF (Harvard Step Test). Predicted age at peak height velocity (APHV) was calculated. Pathway analyses were conducted to identify the influences of the measured variables. Results: APHV harmed the level of ΔFI (fitness index) (β = −0.270; p = 0.035), while HIIT had a positive effect on changes in ΔFI (β = 0.246; p = 0.002). Sex affected the changes in CRF (β = 0.344; p = 0.011). Two models were designed respective to sex. The effect of the APHV on ΔFI in girls was close to significant (p = 0.053) but was non-significant in boys (p = 0.311). Changes in CRF in girls were positively and close to significance (p < 0.100) affected by FMIbaseline. Conclusion: HIIT is an effective way of exerting positive changes in CRF in adolescents, which is greater in boys than girls. APHV plays a role only in girls. The HIIT should be tailored to girls depending on their maturity status.