To more accurately control the morphologies of self-assembled layers of titanium dioxide nanotubes (TNT), 10 turns of high pressure torsion (HPT) was applied to pure titanium to refine the grain size from 13 μm to 135 nm and then two-step electrochemical anodization (30 V, DC, 16 and 6 h) was used to produce TNT layers on these two substrates. Optical microscopy, transmission electron microscopy, scanning electron microscopy and wettability testing showed that grain refinement induced by HPT processing leads to thicker TNT layers with more homogeneous diameters, changed surface morphology and increased aqueous contact angle. The reason why two-step anodization increased the quality of anodized TNT layers and the underlying mechanism for how refined grain size influenced the morphologies and wettability of TNT layers were discussed.