Our objectives were to fabricate three-dimensional collagen/chondroitin sulfate beads using mild conditions and no chemical reagents, and subsequently to investigate the influence of the nanotopography of these beads on primary osteoblast and MG63 cell responses, including cell morphology, proliferation rate, and gene expression. The major principle used to prepare beads was complex coacervation, with which we could obtain a three-dimensional collagen matrix with or without a characteristic D-pattern. Therefore, we utilized primary osteoblasts and MG63, an osteoblast-like cell line, to examine the effects of specific structures on cellular behaviors. We found that the phenotype of primary osteoblasts grown on D-periodic beads was a polygonal shape with extending lamellipodia; however, a greater number of cells displayed a circular morphology on the non-D-periodic carriers. After 7 and 14 days, MG63 cells on D-periodic beads expressed higher levels of osteopontin, alkaline phosphatase activity, type I collagen, and osteocalcin than those on the non-D-periodic beads. Together, these data demonstrate that the unique D-pattern of collagen not only enhances the gene expression and mineralization of osteoblasts but also induces the cells to display the normal phenotype, which indicates that the nanotopography of carriers may regulate cellular responses through the spatial control of downstream signaling.