Gelatin hydrogel (GH) has been proven to be an appropriate carrier for the sustained release of proteins. The W9 peptide (W9) inhibits osteoclastogenesis and accelerates bone morphogenetic protein (BMP)-2-induced ectopic bone formation through type 1 collagen (Col1) as a carrier. The aim of this study was to assess whether GH coupled with W9 elicited improved synergistic effects on BMP-2-induced local bone formation compared to Col1. Methods: The kinetics of W9 release from GH and Col1 were determined using a spectrofluorometer. A calvarial defect model was employed in 5-week-old male C57BL/6 mice (n=24). Each carrier was loaded with BMP-2, BMP-2 + W9, or vehicle, and was applied on the defect site. The mice were sacrificed on day 28 after surgery to create the calvarial defect. Radiographic and bone histomorphometric analyses were performed to assess the healing of the defect. Results: Our in vitro kinetic study showed the sustained release of W9 from the GH compared to Col1. Radiographic analyses of the defect site showed that the combined delivery of BMP-2 and W9 from GH induced higher radio-opacity compared to that induced by combined delivery from Col1. Dual-energy X-ray absorptiometry confirmed these observations. Histomorphometric analyses revealed that the combined delivery of BMP-2 and W9 from GH induced higher bone formation and lower osteoclast number than those related to the delivery from Col1. Conclusions: GH could facilitate the anabolic effects of W9 on BMP-2-induced bone regeneration in a better manner, compared to Col1.z © 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.