Vanadium is a toxic metal listed by the IARC as possibly carcinogenic to humans. Manufactured nanosize vanadium pentoxide (V₂O₅) materials are used in a wide range of industrial sectors and recently have been developed as nanomedicine for cancer therapeutics, yet limited information is available to evaluate relevant nanotoxicity. In this study we used high-resolution metabolomics to assess effects of two V₂O₅ nanomaterials, nanoparticles and nanofibers, at exposure levels (0.01, 0.1, and 1 ppm) that did not cause cell death (i.e., non-cytotoxic) in a human airway epithelial cell line, BEAS-2B. As prepared, V₂O₅ nanofiber exhibited a fibrous morphology, with a width approximately 63 ± 12 nm and length in average 420 ± 70 nm; whereas, V₂O₅ nanoparticles showed a typical particle morphology with a size 36 ± 2 nm. Both V₂O₅ nanoparticles and nanofibers had dose-response effects on aminosugar, amino acid, fatty acid, carnitine, niacin and nucleotide metabolism. Differential effects of the particles and fibers included dibasic acid, glycosphingolipid and glycerophospholipid pathway associations with V₂O₅ nanoparticles, and cholesterol and sialic acid metabolism associations with V₂O₅ nanofibers. Examination by transmission electron microscopy provided evidence for mitochondrial stress and increased lysosome fusion by both nanomaterials, and these data were supported by effects on mitochondrial membrane potential and lysosomal activity. The results showed that non-cytotoxic exposures to V₂O₅ nanomaterials impact major metabolic pathways previously associated with human lung diseases and suggest that toxico-metabolomics may be useful to evaluate health risks from V₂O₅ nanomaterials.