Titanium dioxide (TiO2) nanotube coated substrates have revolutionized the concept of implant in a number of ways, being endowed with superior osseointegration properties and local drug delivery capacity. While accumulating reports describe the influence of nanotube diameter on cell behavior, little is known about the effects of nanotube lateral spacing on cells involved in bone regeneration. In this context, in the present study the MC3T3-E1 murine pre-osteoblast cells behavior has been investigated by using TiO2 nanotubes of ~78 nm diameter and lateral spacing of 18 nm and 80 nm, respectively. Both nanostructured surfaces supported cell viability and proliferation in approximately equal extent. However, obvious differences in the cell spreading areas, morphologies, the organization of the actin cytoskeleton and the pattern of the focal adhesions were noticed. Furthermore, investigation of the pre-osteoblast differentiation potential indicated a higher capacity of larger spacing nanostructure to enhance the expression of the alkaline phosphatase, osteopontin and osteocalcin osteoblast specific markers inducing osteogenic differentiation. These findings provide the proof that lateral spacing of the TiO2 nanotube coated titanium (Ti) surfaces has to be considered in designing bone implants with improved biological performance.