The present study reports the fabrication and characterization of nanofibrous scaffold comprising silk fibroin (SF) and polycaprolactone (PCL) incorporated with vitamin D3 (VD3). The optimized SF/PCL/VD3 nanofibers of average diameter 129±19 nm were obtained by electrospinning technique. The results of the contact angle measurements suggested that the presence of VD3 improves the wettability of the fibers and the water contact angle of the PCL nanofibers decreased from 75±1° to 53±1° after SF and VD3 addition. The elastic modulus for PCL nanofiber was increased from 0.78±0.27 MPa to 2.3±1.16 MPa upon the incorporation of SF and VD3. The cumulative release percentage of VD3 from the scaffolds was 48% during 24 hours. According to MTT assay and cell culture, the nanofibrous scaffolds (SF/PCL/VD3) were superior to PCL and SF/PCL scaffolds in improving the attachment and spreading of adipose derived stem cells (ADSCs), therefore, may be potential candidates for bone tissue engineering applications.