Hyaluronic acid (HyA) is an outstanding new product in the field of oral and maxillofacial surgery. The aim of this study was to evaluate the effects of HyA on bone regeneration in critical-size calvarial defects. Twenty-four female Sprague-Dawley rats were used in the present study. In each rat, 4 critical-size defects received different treatments: no treatment (control); HyA; Graft; and HyA+Graft combination. New bone formation, defect closure, inflammation, vascular proliferation, immature bone formation, mature bone formation, and bone marrow existence were investigated based on histological findings. The healing parameters related to bone formation (new bone formation, defect closure, immature bone formation) were significantly higher in the HyA group compared with the control group. However, HyA alone was unable to induce sufficient bone regeneration compared with treatments involving graft materials (Graft and HyA+Graft). In the Graft and HyA+Graft groups, prominent enhancement of all healing parameters was noted. The present results demonstrate that HyA alone did not adequately enhance bone regeneration in critical-size defects. Moreover, addition of HyA to a biphasic alloplastic graft material did not result in improved regeneration compared with the graft material alone.