Cellular differentiation can be affected by the extracellular environment, particularly extracellular substrates. The nanotopography of the substrate may be involved in the mechanisms of cellular differentiation in vivo. Organelles are major players in various cellular functions; however, the influence of nanotopography on organelles has not yet been elucidated. In the present study, a micropit-nanotube topography (MNT) was fabricated on the titanium surface, and organelle-specific fluorescent probes were used to detect the intracellular organelle organization of MG63 cells. Communication between organelles, identified by organelle-specific GTPase expression, was evaluated by quantitative polymerase chain reaction and western blotting. Transmission electron microscopy was performed to evaluate the organelle structure. There were no significant differences in organelle distribution or number between the MNT and flat surface. However, organelle-specific GTPases on the MNT were dramatically downregulated. In addition, obvious endoplasmic reticulum lumen dilation was observed on the MNT surface, and the unfolded protein response (UPR) was also initiated. Regarding the relationships among organelle trafficking, UPR, and osteogenic differentiation, our findings may provide important insights into the signal transduction induced by nanotopography.