Abstract: Background and Objective: Effectiveness of bone tissue regeneration utilizing cells and scaffold mainly determined by initial seeding density. Initial seeding density controlled cell-cell interaction which greatly influenced cell osteoblast differentiation. Therefore, the objective of this study was to determine the effect of different initial seeding densities on the morphology and osteoblast capability of MC3T3-E1 cells in hydroxyapatite granular scaffold. Materials and Methods: MC3T3-E1 cells at different seeding density, mainly 5×105 and 1×106 cells cm–2 were cultured in the 2-dimensional flask and 3-dimensional granular hydroxyapatite scaffold. Morphology of the cells in both cultures was analyzed using CellB software while biochemical activity was assessed via alkaline phosphatase (ALP) analysis. Results: At both culture conditions, MC3T3-E1 showed a mononucleated, fibroblast-like shape cell with extended cytoplasmic projection. Cells at higher seeding density reached confluency faster within 6 days of culture. Variations in cell seeding density significantly influenced the cell osteodifferentiation as lower initial seeding density resulted in higher ALP activity. This study has shown that the seeded cell population in the 3-dimensional scaffolds clearly affected the degree of osteoblast cell differentiation in which a higher seeding density was not necessarily better. Conclusion: The seeding density played an important role in influencing the corresponding cell differentiation. Therefore, it is preferable to seed cells onto scaffold at optimal lower seeding density as it influenced the corresponding cell osteoblast differentiation.