Our previous research reports that NaOH treatment leads to the formation of a Ti-O-Na titanate layer on the titanium-6-aluminium-4-vanadium (Ti6Al4V) surface. However, this titanium nanosheets (TNS) hydrogel layer is so brittle that it easily detaches from the implant and can cause many problems, including degradation in the living body due to inhomogeneous composition distribution. The aims of the present study were to investigate combined alkali-treatment of Ti6Al4V alloy and then heated and evaluate the ability of this modified surface to affect osteogenic differentiation of rat bone marrow (RBM) cells, to increase the success rate of titanium implants. We fabricated TNS on titanium alloy surfaces by NaOH treatment prior to heat treatment at 600 °C, and determined RBM cell properties and differentiation potential on the surface in comparison to untreated control surfaces. The nanoscale network structures formed by alkali etching markedly enhanced RBM cell adhesion and osteogenic-related gene expression. Other cell behaviors, such as proliferation, alkaline phosphatase activity, osteocalcin deposition and mineralization, were markedly increased on the TNS-modified Ti6Al4V with heat treatment. Our results suggest that titanium implants modified with nanostructures promote osteogenic differentiation, which may improve the biointegration of these implants into the alveolar bone.