Silica nanostructures were fabricated from Pennisetum glaucum (pearl millet) seed husk by acid-pretreatment and calcination. The fabricated silica nanostructure (SN) functional groups, crystalline nature, surface morphology, and particle size were analyzed by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, and transmission electron microscopy, respectively. Additionally, the cytocompatibility of SNs was analyzed on human mesenchymal stem cells (hMSCs) in an MTT assay, propidium iodine (PI) staining, and acridine orange/ethidium bromide (AO/EB) staining. We observed peaks at 1090 and 800 cm-1, which were assigned to symmetric, asymmetric, and bending vibrations of O-Si-O. The SNs showed an amorphous nature with a spherical shape and were 20-60 nm in diameter. The MTT assay results indicated that SNs exhibited cytocompatibility in hMSCs. The PI staining and AO/EB staining results suggested that SNs do not affect nuclear morphology at up to 400 μg/mL. Furthermore, SNs effect on osteogenic differentiation in hMSCs was studied. These results indicate that SNs induced osteogenic differentiation in hMSCs by upregulation of ALP, BSP, ON and RUNX2 genes. Our process could valorize the Pennisetum glaucum agricultural residues to high value products for bone tissue engineering applications.