Background: Deproteinized bovine bone (DBB) is an osteoconductive bone substitute material extensively used in implant dentistry in association with regenerative procedures. A plethora of studies report successful clinical outcomes, including long-term survival and success of dental implants, after DBB grafting in a variety of indications. In order to improve the outcome of healing in terms of amount and/or speed of new bone formation, DBB has often been combined with substances enhancing bone healing. Strontium (Sr) is a substance known to have anabolic effect on bone tissue and is widely used in the treatment of osteoporosis (e.g. Sr ranelate). Recent preclinical in vivo studies indicated that systemic administration of Sr enhances bone regeneration, while coating dental implant with Sr enhances substantially osseointegration. However, whether local administration of Sr may enhance regeneration in bone defects grafted with bone substitute materials has not been evaluated before. Aim/Hypothesis: To histomorphometrically evaluate bone healing after grafting critical-sized calvarial defects (CSD) in rats with Sr-loaded DBB. Material and Methods: Two circular full-thickness bone defects (Ø5 mm) were prepared on each parietal bone of 42 female Wistar rats. The animals were randomly allocated in 3 equal-sized groups, according to the type of biomaterial used to fill one of the two CSD: 1) DBB loaded with 140Μm of Sr per gr biomaterial (DBB/Sr 140); 2) DBB loaded with 700Μm of Sr per gr biomaterial (Group DBB/Sr 700); 3) DBB soaked with sterile saline (DBB). In each treatment group, the other CSD was left empty as negative control). Seven animals in each group were sacrificed after 15 and 60 days from surgery. Histological and histomorphometric evaluation of the tissues contained within the CSD (i.e., % bone, % DBB, % other) was performed on decalcified sections obtained from central aspects of the CSD. Differences among the 3 treatment groups were evaluated with One-way Anova and post-hoc Tukey test, while differences between grafted and non-grafted CSD within each group were evaluated with the paired t-test; the level of significance was set at P < 005. Results: Limited amounts of newly formed bone could be observed in the grafted and non-grafted CSDs, in all 3 groups, after 15 days of healing. After 60 days of healing, CSDs grafted with Sr-loaded DBB showed statistically significant larger relative amounts of newly formed bone comparing to CDSs grafted with DBB soaked in saline (DBB/Sr 140: 8.3 ± 1.1; DBB/Sr 700: 8.7 ± 0.9; DBB: 6.9 ± 00.6). No differences were observed among groups regarding the amount of DBB in any of the experimental periods. Nevertheless, CSDs filled with DBB presented with significant less newly formed bone comparing to the negative controls (14.0–20.2%). Conclusions and Clinical Implications: Grafting of CSDs with Sr-loaded DBB enhanced bone formation comparing to grafting with DBB soaked in saline; however, DBB grafting per se seemed to delay bone formation.