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.