Purpose: This study investigated the effect of enamel matrix derivative (EMD) on spreading, proliferation, and differentiation of osteoblasts cultured on zirconia disks with smooth and rough surfaces. Materials and Methods: EMD was added to the culture medium or coated on zirconia disks that had machined (smooth) or sandblasted (rough) surfaces. The effects of EMD on cell proliferation of MC3T3-E1 osteoblastic cells were examined using a hemocytometer. Osteoblastic differentiation was examined by histologic analysis of alkaline phosphatase (ALP) activity and the degree of mineralization. ALP activity was also measured quantitatively. Scanning electron microscopic analysis was performed to observe cell morphology. Enzyme-linked immunosorbent assay of osteocalcin and reverse-transcriptase polymerase chain reaction of osteocalcin, osteopontin, and type 1 collagen were performed to investigate the expression of osteoblast-related genes. Results: The addition of EMD to the medium enhanced the spreading, proliferation, and differentiation of osteoblasts cultured on zirconia. However, when it was coated on zirconia, EMD reduced osteoblastic spreading and adhesion in the early stage of culture, although it enhanced proliferation and differentiation of osteoblasts in later stages. A promotive effect of EMD on osteocalcin mRNA expression, mineralization, and ALP activity of osteoblasts cultured on the rough surface was observed. Conclusions: EMD may contribute to treatment with zirconia implants via its promotion of osteoblastic proliferation and activity. However, the procedure for application of EMD may be a crucial factor for the outcome of implants. Keywords: dental implants, enamel matrix derivative, osteoblasts, zirconia