To determine whether collagen membrane (CM) fixation enhances guided bone regeneration in standardized defects. Four 8-mm-diameter defects were surgically made in eight rabbit calvaria, and randomly allocated into four groups: control (empty), unfixed-CM, fixed-CM, and unfixed-CM with bone graft (BG + CM) (positive control). After 1- and 4-week healing periods, the animals were sacrificed and quantitative reverse transcription polymerase chain reaction, micro-computed tomography, and histological outcomes were assessed. At week 1, the expression levels of BMP-2, FGF-2, VEGF, and osteocalcin were significantly higher in the fixed-CM group than in the unfixed-CM and control groups (p < .05). Conversely, cathepsin-K was significantly expressed in the unfixed-CM group. No significant differences in expression markers were observed between the fixed-CM and BG + CM groups (p > .05). At week 4, new bone formation was significantly higher in the fixed-CM group than the unfixed-CM and control groups (p < .05), but similar to the BG + CM group (p > .05). CM fixation enhances the expression of osteogenic factors similar to BG + CM, leading to significantly more new bone formation. This suggests that the osteogenic potential is greater when membranes are fixed, thereby limiting the necessity of membrane-supporting materials to enhance bone formation.