Artificial bioactive materials have received increasing attention worldwide in clinical orthopedics to repair bone defects that are caused by trauma, infections or tumors, especially dedicated to the multifunctional composite effect of materials. In this study, a weakly alkaline, biomimetic and osteogenic, three-dimensional composite scaffold (3DS) with hydroxyapatite (HAp) and nano magnesium oxide (MgO) embedded in fiber (F) of silkworm cocoon and silk fibroin (SF) is evaluated comprehensively for its bone repair potential in vivo and in vitro experiments, particularly focusing on the combined effect between HAp and MgO. Magnesium ions (Mg) has long been proven to promote bone tissue regeneration, and HAp is provided with osteoconductive properties. Interestingly, the weak alkaline microenvironment from MgO may also be crucial to promote Sprague-Dawley (SD) rat bone mesenchymal stem cells (BMSCs) proliferation, osteogenic differentiation and alkaline phosphatase (ALP) activities. This SF/F/HAp/nano MgO (SFFHM) 3DS with superior biocompatibility and biodegradability has better mechanical properties, BMSCs proliferation ability, osteogenic activity and differentiation potential compared with the scaffolds adding HAp or MgO alone or neither. Similarly, corresponding meaningful results are also demonstrated in a model of distal lateral femoral defect in SD rat. Therefore, we provide a promising 3D composite scaffold for promoting bone regeneration applications in bone tissue engineering.