We successfully fabricated a three-dimensional (3D) printing-based PCL/PLGA/β-TCP guided bone regeneration (GBR) membrane that slowly released rhBMP-2. To impregnate the GBR membrane with intact rhBMP-2, collagen solution encapsulating rhBMP-2 (5 µg ml(-1)) was infused into pores of a PCL/PLGA/β-TCP membrane constructed using a 3D printing system with four dispensing heads. In a release profile test, sustained release of rhBMP-2 was observed for up to 28 d. To investigate the efficacy of the GBR membrane on bone regeneration, PCL/PLGA/β-TCP membranes with or without rhBMP-2 were implanted in an 8 mm calvaria defect of rabbits. Bone formation was evaluated at weeks 4 and 8 histologically and histomorphometrically. A space making ability of the GBR membrane was successfully maintained in both groups, and significantly more new bone was formed at post-implantation weeks 4 and 8 by rhBMP-2 loaded GBR membranes. Interestingly, implantation with rhBMP-2 loaded GBR membranes led to almost entire healing of calvaria defects within 8 weeks.