Vitamin D and its analogues that are often used in the treatment of osteoporosis have also been shown to affect the healing of bone fractures. To avoid the high-dose systemic treatments, needed to achieve faster healing of fractured bone, the approach of local application of these drugs has become very attractive. The aim of our study was to examine the influence of cholecalciferol and alfacalcidol, locally applied with bovine bone mineral matrix (Bio-Oss), supplemented with platelet-rich plasma on femur defect healing in ovariectomized rats. Experimental osteoporosis was induced by bilateral ovariectomy. To estimate the healing of defects, the formation of the new bone tissue and the resorption of Bio-Oss particles after two and eight weeks of defect healing, we performed biochemical, histological, immunohistochemical, histomorphometric and scanning electron microscopy analyses. Our results showed that locally applied cholecalciferol and alfacalcidol delay early bone healing of defect in ovariectomized rat model. Both of the examined vitamins, particularly alfacalcidol, decreased the resorption of Bio-Oss particles in defects. Only alfacalcidol affected the formation of histologically normal new bone tissue with a good integration into the surrounding osteoporotic bone. Cholecalciferol allowed callus mineralization and led to the formation of new bone with high quantity, with characteristics, similar to those of the control group. Our results suggested that alfacalcidol and cholecalciferol may be promising tools for the local treatment of osteoporotic fractures, but the goal of application should specify the choice of vitamin D form, as well as the composition of scaffolds, with which they are applied.