A number of dermal toxicological studies using TiO2 nanoparticles exist which are based on the study of various animal models like mice, rabbits etc. However, a well-defined study is lacking on the dermal toxic effects of TiO2 nanoparticles on rats, which are the appropriate model for systemic absorption study of nanoparticles. Furthermore, toxicity of TiO2 nanoparticles varies widely depending upon the size, concentration, crystallinity, synthesis method etc. This study was conducted to synthesize TiO2 nanoparticles of different sizes (∼15 to ∼30 nm) by aqueous method, thereby evaluating the concentration-dependent toxicological effects of the ∼20-nm sized nanoparticles on Wistar rats. Characterization of the particles was done by transmission electron microscope, dynamic light scattering instrument, X-ray diffractrometer, and ultraviolet spectrophotometer. The toxicity study was conducted for 14 days (acute), and it is observed that TiO2 nanoparticles (∼20 nm) at a concentration of 42 mg/kg, when applied topically showed toxicity on rat skin at the biochemical level. However, the histopathological studies did not show any observable effects at tissue level. Our data suggest that well-crystallized spherical-shaped ∼20 nm anatase TiO2 nanoparticles synthesized in aqueous medium can induce concentration-dependent biochemical alteration in rat skin during short-term exposure.