Background: Gene-activated matrix (GAM) induces sustained local production of growth factors to promote tissue regeneration. GAM contains a plasmid DNA (pDNA) encoding target proteins that is physically entrapped within a biodegradable matrix carrier. GAM with a pDNA encoding the first 34 amino acids of parathyroid hormone (PTH 1–34) and a collagen matrix enhances bone regeneration in long bone defects. Demineralized freeze-dried bone allograft (DFDBA) is a widely used osteoinductive bone graft. The present study determined the osteogenic effects of PTH-GAM with a collagen or DFDBA/collagen composite (D/C) matrix for treating craniofacial bone defects. Methods: We constructed a pDNA encoding human PTH 1–34 and performed cyclic AMP ELISA to verify the bioactivity of PTH 1–34. Next, we generated a D/C matrix and PTH-GAMs containing a collagen matrix (PTH-C-GAM) or D/C matrix (PTH-D/C-GAM). Rats with critical-sized calvarial bone defects were divided into four groups, namely, untreated rats (sham group) and rats grafted with D/C matrix, PTH-C-GAM, or PTH-D/C-GAM (D/C, PTH-C-GAM, or PTH-D/C-GAM groups, respectively). PTH expression was determined by performing immunohistochemical staining after 4 and 8 weeks. New bone formation was evaluated by performing radiography, dual-energy X-ray absorptiometry, microcomputed tomography, and histological examination. Results: PTH pDNA-transfected cells secreted bioactive PTH 1–34. Moreover, PTH was expressed at 4 and 8 weeks after the surgery in rats in the PTH-C-GAM group but not in rats in the D/C group. New bone formation in the calvarial bone defects, from more to less, was in the order of PTH-D/C-GAM, PTH-C-GAM, D/C, and sham groups. Conclusion: Our results indicate that PTH-GAM with a collagen matrix promotes local PTH production for at least 8 weeks and bone regeneration in craniofacial bone defect. Moreover, our results indicate that replacement of the collagen matrix with the D/C matrix improves the osteogenic effects of PTH-GAM.