Restoration of the lost bone volume is one of the most deliberate issues in dentistry. Sustained-release microspherical oxytocin hormone in a poloxamer hydrogel scaffold combined with a mixture of beta-tricalcium phosphate and hydroxyapatite (CP) may serve as a suitable bone graft. The aim of this study was to design and test a novel thermosensitive hydrogel graft incorporating oxytocin-loaded poly(d, l-lactide-co-glycolide) (PLGA) sustained-release microspheres and CP. Thermosensitive poloxamer hydrogel containing CP (HCP graft) was prepared as a base and combined with hollow microspheres (HCPM) and oxytocin-loaded microspheres (HCPOM). Eighty Wistar rats were used for testing the grafts and a control group in 8-mm-diameter critical-sized calvarial defects (CSD); (n = 20). Bone healing at the 4th and 8th weeks was evaluated by histological, histomorphometric, and radiological (micro-computed tomography [mu CT]) analyses. The results were analyzed by two-way analysis of variance (P < .05). Oxytocin-loaded PLGA microspheres prepared by the solvent displacement method yielded a high encapsulation efficiency of 89.5% and a slow drug release. Incorporation of the microspheres into the hydrogel graft slowed the release rate down and the release completed within 32 days. HCPOM revealed the highest new bone formation (26.45% +/- 6.65% and 30.76% +/- 4.37% at the 4th and 8th weeks, respectively; P < .0001) while HCPM and HCP groups revealed a bone formation of around 10% (P > .05). mu CT findings of HCPOM group showed the highest mean bone mineral density values (42.21 +/- 5.14 and 46.94 +/- 3.30 g/cm(3) for the 4th and 8th weeks, respectively; P < .0027). The proposed oxytocin-loaded sustained-release PLGA microspheres containing thermosensitive hydrogel graft (HCPOM) provide an accelerated bone regeneration in the rat calvaria.