The bone-like self-organized hydroxyapatite/collagen (HAp/Col) nanocomposite sponge was prepared from HAp/Col nanocomposite fibers. We analyzed osteogenic activity of human osteoblastic MG63 cells in the HAp/Col sponge under pressure/perfusion culture. Collagen (Col) sponge was used as a control. The MG63 cells were attached well and showed good proliferation in the HAp/Col sponge. The total DNA content in the HAp/Col sponge was approximately 1.8 times greater than that in the Col sponge at 21 days. The MG63 cells showed good osteogenic gene expression in the HAp/Col sponge by reverse transcription-polymerase chain reaction analysis. These result suggested that HAp/Col sponge can be useful for bone tissue engineering scaffold materials.