Nanostructured titanium dioxide (TiO₂) semiconductor materials have significant applications in photocatalysis and photovoltaics. In this study, a biomimetic approach was developed to fabricate nanostructured TiO₂ that possessed hierarchical structure using diatom shells as a template. Diatoms are single-celled algae that make silica shells called frustules that possess periodic structures ordered at the micro- and nanoscale. Biosilica frustules were isolated from cell cultured Pinnularia diatom by hydrogen peroxide treatment of cell biomass. Poly-L-lysine (PLL) was conformally adsorbed onto surface of the frustule biosilica. The condensation of soluble Ti-BALDH to nanostructured TiO₂ by PLL-adsorbed diatom biosilica loaded 0.8 g Ti / g Si onto the frustule. TiO₂ nanoparticles preferentially deposited into the 200 nm frustule pores and also coated the frustule outer surface. The periodic pore array templated these concentrated pockets of TiO₂ nanoparticles into an ordered, lattice-like structure. Thermal annealing at 680 ^oC converted the as-deposited TiO₂ to its anatase form with an average nanocrystal size of 19 nm, as verified by XRD, electron diffraction, and SEM/TEM. This is the first reported study of directing the peptide-mediated deposition of TiO₂ into a hierarchical nanostructure using a biologically fabricated template.