The aim of this study was to evaluate the effects of local administration of human amniotic fluid (HAF) on newly formed bone obtained by mandibular distraction osteogenesis (DO) with histomorphometry. A unilateral mandibular osteotomy at the left corpus was performed in 32 adult male rabbits. After a 5-day latency period, the left mandibles were lengthened by mandibular DO over 5 days, at a rate of 1 mm/day, via a custom-made distractor. After the distraction, the rabbits were divided randomly into four groups: 0.3 ml HAF was injected into the distraction gap followed by 21 (group 1) or 45 (group 2) days of consolidation; or 0.3 ml normal saline (NS) was administered followed by 21 (group 3) or 45 (group 4) days of consolidation. Mandibles were removed at the end of the consolidation period and investigated histomorphometrically. The newly formed bone area (NFBA) and number of fibroblasts increased significantly in the HAF groups compared to the NS groups (NFBA: group 1 vs. group 3, P < 0.05; group 2 vs. group 4, P < 0.01; fibroblasts: group 1 vs. group 3, and group 2 vs. group 4, P < 0.05), and also in both 45-day consolidation groups compared to the 21-day consolidation groups (NFBA: group 1 vs. group 2, and group 3 vs. group 4, P < 0.001; fibroblasts: group 1 vs. group 2, and group 3 vs. group 4, P < 0.01). Additionally, the numbers of osteoblasts and capillaries were increased significantly at 45 days of consolidation compared to 21 days in both the HAF and NS groups (osteoblasts: group 1 vs. group 2, P < 0.01; group 3 vs. group 4, P < 0.05; capillaries: group 1 vs. group 2, and group 3 vs. group 4, P < 0.01). Histomorphometric analysis demonstrated that local HAF administration effectively accelerated bone formation. Thus, a HAF injection procedure could improve new bone formation around the bone in maxillofacial operations such as DO.