Biomaterials are extensively used in bone defect recovery caused by bone diseases. Multi-walled carbon nanotubes have been reported to reinforce synthetic polymeric materials. The aim of the study is to test poly(3-hydroxybutyrate-co-3-hydroxyvalerate) loaded with different amounts of multi-walled carbon nanotubes to fabricate nanocomposites. Mechanical, mineralization, and degradation properties were studied in vitro. The proliferation and differentiation of rat bone marrow stem cells were studied to determine biocompatibility in vivo. The incorporation of multi-walled carbon nanotubes greatly increased the mechanical properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and the strongest composite obtained was at 2% multi-walled carbon nanotubes. The 2% nanocomposite also had higher rat bone marrow stem cell adhesion, proliferation, and differentiation characteristics compared to the pure poly(3-hydroxybutyrate-co-3-hydroxyvalerate). The apoptosis in the later stage of rat bone marrow stem cells decreased in the 2% nanocomposites group at different time points. Based on histology and micro-computed tomography tests 6 weeks after in vivo implantation, the 2% multi-walled carbon nanotubes/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) treated animals had a higher volume of bone formation compared to the pure poly(3-hydroxybutyrate-co-3-hydroxyvalerate) group. Thus, the presence of multi-walled carbon nanotubes has an apparent positive effect on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in assisting osteogenesis.