The adhesion of cells to surfaces, as well as their proliferation, migration, and differentiation, is guided not only by chemical functionalization but also by surface nanostructuring, nanotopography. [1] Nano-patterned titanium surfaces are one example in which the scale of patterning controls the size of focal adhesions. [2] Nanoscale disorder in surface structure can be used to stimulate cell differentiation [3] or can also be used to maintain stem cell phenotypes over long times. [4] Nanoroughness modulates cells interactions and function via mechanosensing. [5] These all suggest that the careful control of surface nanostructure of such important as titanium (Ti) biomaterial [6] could be a useful tool to achieve desired cellular responses.We first time highlight that ultrasonic treatment is able to produce surface porous sponge layer in Ti. We find it great technological advances that Ti can be also modified with highintensity ultrasonic treatment. We really think that presenting high-intensity ultrasonic technique for Ti nanostructuring increases interest of scientists to the technique. Great advantage of our methodology is a large number of synthetic parameters which can be optimized to tune surface nanostructuring in a controllable manner. Moreover, this methodology will be very interesting in future to provide single-step hybrids and effective loading of porous structures with active chemicals. We compare our methodology with more known for bio-application anodization for surface nanostructuring.Anodization leads to TiO 2 nanotube arrays covering the surface of titanium and is one of the most studied methods to develop porous surface nanotopographies with controlled pore sizes. [6] It has been demonstrated in cell culture experiments on TiO 2 nanotube arrays of different sizes that adhesion, proliferation, and migration of mesenchymal stem cells are optimal on ordered nano-pore arrays with spacings in the range of 15-30 nm; these length scales also lead to significantly less apoptosis than on 100 nm structures. [7,8] It should be taken into account that nanostructuring does affect cell function at many levels but in a cell specific manner, and smaller surface features (50 nm) tend to favor cell proliferation in comparison to larger features (300 nm). [9] Anodization requires a conductive substrate, is difficult to use over large surface areas, and uses aggressive media for synthesis. [6] Recently, we have shown that ultrasonic treatments in aqueous media can produce surface porosities in various size-ranges also below 100 nm in metals such as Al or Mg. [10] Here, we demonstrate that such nanostructuring can also be effectively induced in an important biomaterial, titanium, by investigating its influence on cell behavior in comparison to the well-established electrochemical method.To this end, we investigate the response in terms of morphology, adhesion, proliferation, and differentiation of C2C12 cells on a glass substrate and on three different titanium/TiO 2 surfaces: a titania mesoporous sponge layer (...