In vitro propagation of osteoblasts in three-dimensional culture has been explored as a means of cell line expansion and tissue engineering purposes. Studies investigating optimal culture conditions are being conducted to produce bone-like material. This study demonstrates the use of collagen microcarrier beads as a substrate for three-dimensional cell culture. We have earlier reported that microcarriers consisting of cross-linked type I collagen support chondrocyte proliferation and synthesis of extracellular matrix. In this study, we investigated the use of collagen microcarriers to propagate human trabecular bone-derived osteoblasts. Aggregation of cell-seeded microcarriers and production of extracellular matrix–like material were observed after 5 d in culture. Expression of extracellular matrix proteins osteocalcin, osteopontin, and type I collagen was confirmed by messenger ribonucleic acid analysis, radioimmunoassay, and Western blot analysis. The efficient recovery of viable cells was achieved by collagenase digestion of the cell-seeded microcarriers. The collagen microcarrier spinner culture system provides an efficient method to amplify large numbers of healthy functional cells that can be subsequently used for further in vitro or transplantation studies.