The aim of the present in vivo microcomputed tomographic (μCT), histologic, and biomechanical study was to assess the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) for promoting guided bone regeneration (GBR) in a standardized rat calvarial defect model. Forty female Wistar albino rats with a mean age of 7.5 months and mean weight of 275 g were used. Following calvarial exposure under general anesthesia, a full-thickness standardized calvarial defect (4.6 mm in diameter) was created. The study animals were randomly divided into four groups based on biomaterials used for GBR: (1) no treatment (negative control); (2) bone graft alone; (3) bone graft placed in the defect and covered with a collagen membrane (CM); and (4) bone graft soaked in BMSCs and covered with a CM. Bone volume and bone mineral density (BMD) of newly formed bone (NFB) and remnant bone particles were determined at baseline and at 2, 4, 6, 8, and 24 weeks postoperative using real time in vivo μCT. Histologic and biomechanical analyses of calvarial specimens were performed at 24 weeks, when the rats were euthanized. Statistically significant differences in volume and BMD of NFB were observed between and within the groups at different data collection periods. Significant increases in volume and BMD of NFB occurred as early as week 2 in all groups except the negative control. While the greatest volume of NFB was observed in the bone graft + BMSC + CM group, BMD of NFB was significantly higher in the bone graft + CM group. Statistically significant decreases in volume and BMD of remnant bone particles were also observed between the groups. Histologic analysis revealed NFB in all groups. The hardness and elastic modulus of NFB in the bone graft + BMSC + CM group were significantly higher than that in the other groups and also similar to adjacent natural bone. This study shows that using adjunct BMSCs with bone graft and CM for guided bone regeneration in standardized rat calvarial defects resulted in the highest quality and quantity of NFB.