N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-quinone) is an emerging contaminant of concern that is generated through the environmental oxidation of the rubber tire anti-degradant 6PPD. Since the initial report of 6PPD-quinone being the cause of urban runoff mortality syndrome of Coho salmon, numerous species have been identified as either sensitive or insensitive to acute lethality caused by 6PPD-quinone. In sensitive species, acute lethality might be caused by uncoupling of mitochondrial respiration in gills. However, little is known about effects of 6PPD-quinone on insensitive species. Here we demonstrate that embryos of fathead minnows (Pimephales promelas) are insensitive to exposure to concentrations as great as 39.97 μg/L for 168 h, and adult fathead minnows are insensitive to exposure to concentrations as great as 9.4 μg/L for 96 h. A multi-omics approach using a targeted transcriptomics array, (EcoToxChips), and proton nuclear magnetic resonance (1H NMR) was used to assess responses of the transcriptomes and metabolomes of gills and livers from adult fathead minnows exposed to 6PPD-quinone for 96 h to begin to identify sublethal effects of 6PPD-quinone. There was little agreement between results of the EcoToxChip and metabolomics analyses, likely because genes present on the EcoToxChip were not representative of pathways suggested to be perturbed by metabolomic analysis. Changes in abundances of transcripts and metabolites in livers and gills suggest that disruption of one‑carbon metabolism and induction of oxidative stress might be occurring in gills and livers, but that tissues differ in their sensitivity or responsiveness to 6PPD-quinone. Overall, several pathways impacted by 6PPD-quinone were identified as candidates for future studies of potential sublethal effects of this chemical.