Electrochemical treatments, such as electropolishing on titanium alloys, are used to promote the formation of nanostructured surfaces, which can contribute to the bone regeneration process. However, sterilization methods can change the superficial and physicochemical properties of the biomaterials. The objective of this work was to evaluate the effect of three sterilization methods (air plasma, ethanol + PBS, and autoclave sterilizations) on nanostructured Ti6Al4V surfaces properties obtained by electrochemical treatment. These methods, especially the first two, have been widely used in literature, yet few studies in the literature highlight the changes on the surface of the samples. The nanostructures were obtained by electropolishing in a H2SO4/HF/glycerine solution, at 25 V and at 7 °C for 4 min. Samples were characterized by atomic force microscopy (AFM), profilometry, and wettability. Samples were seeded with hESC-MPs, and the cell number was measured. The air plasma sterilization did not promote changes in nanometric morphology and roughness of the Ti6Al4V nanostructured samples. Unlike air plasma sterilization, the ethanol + PBS and the autoclave sterilizations, which strongly affected the nanostructured surface morphology and properties, and, consequently, the cellular viability after 7 days of contact with human embryonic stem cell-derived mesenchymal progenitors (hESC-MPs).