The purpose of this pilot study was to evaluate for the first time the effect of 75/25 w/w nano-Hydroxyapatite/Chitosan (nHAp/CS) scaffolds on Guided Bone Regeneration (GBR) in rat calvarial critical-sized defects (CSDs). Six adult Sprague Dawley rats, 3 males and 3 females, were used. Two CSDs, full thickness and 5mm in diameter, were trephined in both sides of the parietal bone. The right CSD was filled with nHAp/CS scaffold, while the left CSD remained empty, as the control group. The wound was sutured in layers. Rats were euthanized with diethyl ether inhalation at 2, 4 and 8 weeks after surgical procedure. Histological and histomorphometric analysis was performed within distinct regions of interest (ROI): the lateral area inward of the middle sagittal seam; the lateral area outward of the middle sagittal seam and the central area. The mean surface of newly formed bone (in ?m2) in the lateral area inward of the middle sagittal seam of all rats was significantly higher (P=0.039) in the experimental group (91733.00±38855.60) than the control group (46762.17±25507.97). The NOex-c, defined as total number of osteocytes (OST) in newly formed bone surface in experimental group [experimental OST] minus the total number of osteocytes in newly formed bone surface in control group [control OST], was significantly greater (P=0.029) at 4th week post-surgery. Within the experimental group, a statistically significant increase (P=0.042) in the surface of newly formed bone was noticed in rats euthanized in 4th week compared with rats euthanized in 2nd week after surgery in the lateral area inward of the middle sagittal seam. The results of this study suggest that 75/25 w/w nHAp/CS scaffolds should be considered as a suitable biomaterial for GBR.