Ethionamide (ETA) is the most frequently prescribed oral second-line anti-tuberculosis drugs in children at present. The drug undergoes extensive metabolism by flavin-containing monooxygenase isoform 3 (FMO3) in the liver to exert its cytotoxic effects. In vivo, a significant age-related change in ETA elimination has been observed when comparing different age groups in children. However, so far, the exact mechanism of this dynamic increase has not been characterized. To address this issues, we hypothesized the age-dependent changes in ETA pharmacokinetics (PKs) are mostly due to the progressive increases in expression and metabolic capacity of FMO3 during childhood. In order to test the hypothesis, a full physiologically based pharmacokinetic (PBPK) model of ETA was established and validated in adults by incorporating comprehensive metabolism and transporter profiles and subsequently expanded to the pediatric population by integrating FMO3 maturational development over time. As a result, a well-predicted PBPK model was validated successfully both in adults and pediatrics that was then applied to determine the critical role of FMO3 in the mechanistic elimination pathway of ETA through quantification of its contribution to ETA pharmacokinetic. Additionally, a significant correlation between the systemic clearance and age was observed in children by consideration of FMO3 maturation which could offer the mechanistic understanding of age-dependent changes of ETA elimination. In conclusion, this study based on the principle of in vitro-in vivo extrapolation demonstrated the FMO3 ontogeny in children should be taken into account in ETA prescribing information to help drive personalized medicine in tuberculosis treatment