The purpose of this study was to investigate the effect of different bone graft substitutes onto the growth and proliferation pattern of bone cells derived from human iliac cancellous bone. Five different bone graft materials were used to investigate the effect on the proliferation of osteoblasts in vitro: phytogene hydroxyapatite (Algipore), alpha-Tricalcium phosphate (Bio-Base), bovine hydroxyapatite (low temperature) (Bio-Oss), bovine hydroxyapatite (high temperature) (Osteograf), and bovine hydroxyapatite (high temperature) enhanced with p-15, synthetic peptide (PepGen p-15). The osteoblasts were derived from human iliac cancellous bone and seeded with the different bone substitutes. The cell proliferation and viability (WST-1), alkaline phosphatase as an early marker of osteoblast proliferation, was evaluated after 6 and 9 days. The cultures were examined for cell growth pattern and morphology by normal light and scanning electron microscopy. The human osteoblasts showed a different proliferation pattern according to the type of applied bone graft substitute. PepGen P-15 showed the highest proliferation and differentiation rate followed by Osteograf, Algipore, and Bio-base. Bio-Oss showed the lowest. These results were confirmed by electron microscopy and light microscopy evaluation in which similar growth pattern were observed. Distinct bone graft materials have different impact onto the proliferation pattern of human osteoblasts in vitro.