The large bone defect often require bone transplant or its substitutes to restore bone integrity which have some limitations. The study was conducted to analyze role of autologous osteoblast that are amplified in vivo and impregnated in a synthesized three-dimensional gelatin hydroxyapatite scaffold for treating artificially created critical size defect in rabbit's iliac crest. In this research, 4-month-old ten healthy white male rabbits of average weight 2-3kg were chosen. Osteoblasts cells were isolated from the rabbit iliac crest and were taken in transfer medium to the laboratory and cultured for 2-3weeks. These osteoblast cells were seed on 3-dimensional scaffold and culture the construct for 2weeks. The cultured autologous osteoblasts over the scaffold were transplanted into the defect by reopening the iliac crest of the same rabbit from which is cells were extracted. Serial radiograph of pelvis was done to see progressive signs of union. Out of ten rabbits chosen for study two rabbits were passed during study. Gross and radiological examination of rabbits was done at 0, 4, 8 and 12weeks. Features of union was seen in six rabbits on follow-up. There is no signs of union or minimal new bone formation around the implant material were seen in two case. The study demonstrated using autologous cultured osteoblasts impregnated over three-dimensional biodegradable scaffold for large bone defect is a good option. The importance of three-dimensional biodegradable scaffold is that it provide scaffolding for sufficient interval for new bone formation.