As titanium dioxide (TiO2) nanoparticles are widely used commercially, their potential biosafety and metabolic mechanism needs to be fully explained. In this study, the cytotoxicity of homogeneous and weakly aggregated (< 100 nm) TiO2 nanoparticles was investigated by analyzing the changes in metabolite profiles both in mouse fibroblast (L929) cells and their corresponding culture media using gas chromatograph with a time-of-flight mass spectrometry (GC/TOFMS)-based metabolomic strategy. With multivariate statistics analysis, satisfactory separations were observed in principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) models. Based on the variable importance in the OPLS-DA models, a series of differential metabolites were identified by comparison between TiO2 nanoparticle-treated L929 cells or their corresponding culture media and the control groups. It was found that the major biochemical metabolism (carbohydrate metabolism) was suppressed in TiO2 nanoparticle-treated L929 cells and their corresponding culture media. These results might account for the serious damage to energy metabolism in mitochondria and the increased cellular oxidation stress in TiO2 nanoparticle-induced L929 cells. These results also suggest that the metabolomic strategy had a great potential in evaluating the cytotoxicity of TiO2 nanoparticles and thus was very helpful in understanding its underlying molecular mechanisms. Copyright © 2012 John Wiley & Sons, Ltd.