The stability of orthopedic implants is governed by the interaction between the material's surface and bone cells. Nanosized structures seem to be promising in supporting the osseointegration of total joint replacements. In order to analyze whether nanostructured titanium surfaces, as generated by a short‐pulsed Nd:YVO4 laser irradiation process developed in the aeronautics and space industry as a “cold ablation” process, can be useful for orthopedic applications, their impact on bone cells is evaluated in vitro. Cell spreading and morphology on nanostructured titanium niobium nitride or titanium plasma‐sprayed surfaces are comparable to osteoblast behavior on corresponding untreated titanium or cells grown on cover glass. However, on the nanostructured surfaces cell numbers appear to be reduced. Nonetheless, cell viability is not affected by laser pretreatment. Interestingly, osteoblast proliferation on nanostructured titanium is inhibited to the benefit of an increased production of osteoblast‐specific proteins such as bone alkaline phosphatase, osteocalcin, or procollagen. Furthermore, an enhanced mineralization of cells grown on nanostructured surfaces is confirmed in terms of an elevated hydroxyapatite deposition. Laser irradiation leads to an additional nanostructure on microstructured titanium surfaces and might positively affect the biocompatibility and ingrowth of prostheses made from the corresponding biomaterials.