The influence of silicon-substituted hydroxyapatite (Si-HAp) on osteogenic differentiation was assessed by biological analysis. Si-HAp was prepared by ultrasonic spray pyrolysis (USSP) technique using various amounts of Si (0, 0.8, and 1.6 mass%). Chemical analysis revealed that Si was incorporated into the hydroxyapatite (HAp) lattice with no other crystalline phase and which caused the change of crystal structure. Biological analyses showed that the Si contents affected the cell proliferation and morphology, suggesting that there is an optimal Si content for cell culture. As for differentiation, alkaline phosphatase activity and osteocalcin production of Si-HAp were higher than those of HAp. Gene expression profiles also revealed that substitution of Si (0.8 mass%) up-regulated the expression levels of osteocalcin and especially Runx2, a master gene for osteoblast development. These results suggest that incorporating Si into the HAp lattice may enhance the bioactivity, particularly during early osteoblast development.