Thin films of 3D-nanoflowers of rutile TiO₂ on conducting (FTO and ITO) and non-conducting (glass) substrates were grown using a surfactant free one-step hydrothermal process. Field emission scanning electron microscopy (FE-SEM) observations confirmed the transformation of TiO₂ nanostructures from mesh-like to 3D-nanoflowers with an increase in hydrolysis rate during the growth of the TiO₂ films. The X-ray diffraction (XRD) pattern of the TiO₂ nanostructures as films grown on different substrates showed that under various conditions, they have a phase pure rutile crystallite structure. The high resolution transmission electron microscopy (HR-TEM) diffraction pattern of the TiO₂ nanostructures showed tightly packed assemblies of titanium atoms and a lattice spacing of 0.23 nm along the longitudinal axis direction of rutile TiO₂. X-ray photoelectron spectroscopic (XPS) analysis of the TiO₂ nanostructures grown as films on glass substrates showed a spectral shift of 0.53 eV in binding energy, which confirms the charge accumulation on the non-conducting substrate, whereas there was no spectral shift observed for TiO₂ films with similar structures grown on conducting substrates. The accumulated charge on the conducting surfaces can be easily neutralized, whereas the non-conducting surfaces may retain these accumulated charges. The adhesion, viability and proliferation response of mouse osteoblast (MC3T3) and human stromal (HS-5) cells on the 3D-nanoflowers of TiO₂ as films grown on conducting and non-conducting substrates were assessed. The adhesion and proliferation of both the type of cells showed a better response on non-conducting surfaces as compared to conducting surfaces, despite the similar crystallite structures and nanomorphology of TiO₂. Stromal cells had potential to prepare extra-cellular matrix scaffolds for the ex vivo expansion/differentiation of stem cells. Therefore, the current findings can be used to prepare 3D TiO₂ nanostructure supported cellular scaffolds for regenerative medicine in the future.