Distraction osteogenesis (DO) is widely utilized for treating limb length discrepancy, nonunion, bone deformities and defects. This study sought to develop a 4D time-lapse morphometry method to quantify bone formation and resorption in mouse femur during DO based on image registration of longitudinal in vivo micro-CT scans. Female C57BL/6 mice (n = 7) underwent osteotomy, followed by 5 days of latency, 10 days of distraction and 35 days of consolidation. The mice were scanned with micro-CT at Days 5, 15, 25, 35, 45, and 50. Histological sectioning and Movat Pentachrome straining were performed at Day 50. After registration of two consecutive micro-CT images of the same bone (day x and day y), the spatially- and temporally-linked sequences of formation, resorption and quiescent bones at the distraction gap were identified and bone formation and resorption rates (BFR_dayx-y and BRR_dayx-y) were calculated. The overall percentage error of the registration method was 2.98% ± 0.89% and there was a strong correlation between histologically-measured bone area fraction and micro-CT-determined bone volume fraction at Day 50 (r = 0.89, p < 0.05). The 4D time-lapse morphometry indicated a rapid bone formation during the first 10 days of the consolidation phase (BFR_day15–25 = 0.14 ± 0.05 mm³/day), followed by callus reshaping via equivalent bone formation and resorption rates. The 4D time-lapse morphometry method developed in this study allows for a continuous quantitative monitoring of the dynamic process of bone formation and resorption following distraction, which may offer a better understanding of the mechanism for mechano-regulated bone regeneration and aid for development of new treatment strategies of DO.