The unique physico-chemical properties of nano crystalline anatase titanium dioxide nanoparticles (TiO NPs) render them with different biological and chemical activities. Hence, it is widely used in industrial and consumer applications. Previous studies have shown the genotoxicity of TiO NPs. However, there is a paucity of data regarding mutagenicity of these NPs. In the present study, the cellular uptake, sub-cellular localization, cytotoxicity and short term DNA interaction of TiO NPs (1-100 μgmL) of diameter ranging from 12 to 25 nm on mammalian lung fibroblast cells (V-79) has been studied. The flow cytometric analysis and electron micrographs of V-79 monolayer showed the internalization of TiO NPs in the cytoplasm with the confirmation of elemental composition through SEM/EDX analysis. TEM analysis also showed TiO NPs induced ultra-structural changes such as swollen mitochondria and nuclear membrane disruption in V-79 cells. TiO NPs generated free radicals, which induced indirect mutagenic and genotoxic responses. Apart from measuring the genotoxicity by Comet assay, the mutagenic potential of TiO NPs in V-79 cells was evaluated by mammalian HGPRT gene forward mutation assay, showing a 2.98- fold increase in 6TG HGPRT mutant frequency (*p < 0.05, **p < 0.01, ***p < 0.001) by culture plate method, which is an early indicator of potential carcinogenicity. Hence, TiO NPs should be closely monitored and there should be a judicious use and disposal of NPs.