The increasing use of nanotechnology in our daily life can have many unintended effects and pose adverse impact on human health, environment and ecosystems. Wider application of engineered nanoparticles, especially TiO2 nanoparticles (TiO2 NP) necessitates the understanding of toxicity and mechanism of action. Metabolomics provides a unique opportunity to find out biomarkers of nanoparticles exposure, which leads to the identification of cellular pathways and their biological mechanisms. Gas chromatography mass spectrometry (GC–MS)-based metabolomics approach was used in the present study to understand the toxicity of sub-lethal concentrations (7.7 and 38.5 µg/ml) of TiO2 NP (<25 nm) in well-known, soil nematode Caenorhabditis elegans (C. elegans). Multivariate pattern recognition analysis reflected the perturbations in the metabolism (amino acids, organic acids, sugars) of C. elegans on exposure to TiO2 NP. The biological pathways affected due to the exposure of TiO2 NP were identified, among them mainly affected pathways are tricarboxylic acid (TCA) cycle, arachidonic acid metabolism and glyoxalate dicarobxylate metabolism. The manifestation of differential metabolic profile in organism exposed to TiO2 (NP or bulk particle) was witnessed as an effect on reproduction. The present study demonstrates that metabolomics can be employed as a tool to understand the potential toxicity of nanoparticles in terms of organism–environment interactions as well as in assessing the organism function at the molecular level.