Repairing critical-sized bone defects has been a major challenge for orthopedic surgeons in the clinic. The generation of functioning bone tissue scaffolds using osteogenic induction factors is a promising method to facilitate bone healing. In the present study, three-dimensional (3D) printing of a poly(lactic-co-glycolic acid) (PLGA) scaffold with simvastatin (SIM) release functioning was generated by rapid prototyping, which was incorporated with collagen for surface activation, and was finally mixed with SIM-loaded PLGA microspheres. In vitro assays with bone marrow-derived mesenchymal stem cells were conducted. For the in vivo study, scaffolds were implanted into segmental defects created on the femurs of Sprague-Dawley rats. At 4 and 12 weeks following surgery, X-ray, micro-computed tomography and histological analysis were performed in order to evaluate bone regeneration. The results demonstrated that collagen functionalization of PLGA produced better cell adhesion, while the sustained release of SIM promoted greater cell proliferation with no significant cytotoxicity, compared with the blank PCL scaffold. Furthermore, in vivo experiments also confirmed that SIM-loaded scaffolds played a significant role in promoting bone regeneration. In conclusion, the present study successfully manufactured a 3D printing PLGA scaffold with sustained SIM release, which may meet the requirements for bone healing, including good mechanical strength and efficient osteoinduction ability. Thus, the results are indicative of a promising bone substitute to be used in the clinic.