The aim of this study was to assess the response of preosteoblasts (SV-HFO), from adhesion to matrix mineralization, on Ti6Al7Nb surfaces modified by plasma electrolytic oxidation (PEO). Two different surfaces have been generated by changing the oxidation duration from 1 to 5 minutes. The resultant (PEO1 and PEO5) surfaces showed uniform porous topographies with pores mostly in the submicron range and a mixture of anatase and rutile TiO2 phases. However, the average surface roughness, maximum peak-to-valley height, pore size and Ca/P ratio increased with oxidation time whereas pore density and surface porosity decreased. On the PEO1 surface SV-HFO cells attached and spread easily using the pores as anchoring sites for their extensions and showing cell-cell contact after 48 hours. The larger pores protruding from the PEO5 surface suppressed cell adhesion. Deposition of extracellular matrix started earlier on the PEO1 surface and after 21 incubation days a net-like structure well integrated with the porous surface was visible. Matrix mineralization was evidenced on both surfaces after 21 days. However, more uniform mineralized areas were observed on the PEO1 surface whereas an accelerated mineralization was noticed after 14 days on the PEO5 surface. In conclusion, by varying only one PEO process condition, significant changes occurred on the surface of Ti6Al7Nb alloy, which influenced both the early and late response of SV-HFO cells. The observed surface-induced effects indicated that surfaces produced at shorter oxidation time may be more beneficial for early osteogenesis.