Tire wear particles are a pervasive form of microplastics in the environment, produced from the abrasion between vehicle tires and road surfaces. Leachates from tire wear particles can introduce pollutants into the environment. One compound that has been detected in multiple environmental matrices is N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), an additive that is ubiquitously used in tire production to increase product lifetime. Since most tire wear particle emissions are deposited in soil, 6PPD may leach out and pose a potential risk to soil organisms. We studied the acute toxicity of 6PPD on earthworms (Eisenia fetida) by investigating both traditional toxicity and molecular-level endpoints. The acute median lethal concentration (LC50) was 693 μg/g, following a 14-day exposure period. Targeted mass spectrometry-based metabolomics was employed to measure the metabolic responses of earthworms exposed for 48h to five 6PPD concentrations (0.12, 1.2, 12, 120, and 1,200 μg/g). At the highest concentration, multivariate analysis showed that changes to the metabolic profile of earthworms were statistically significant (p = 0.006) compared to the unexposed group. Furthermore, normalized concentrations of alanine, which has been reported as universal bioindicator of oxidative stress, were significantly upregulated with high concentrations of 6PPD exposure. Biochemical pathway analyses revealed disruptions to protein synthesis, amino acid metabolism, and energy metabolism. No metabolites were significantly perturbed after exposure to 6PPD concentrations 0.12-120 μg/g, compared to the control (p > 0.05), suggesting that sorption may have reduced the bioavailability and impact of 6PPD on earthworms based on its octanol-water partition coefficient (log KOW = 4.47). This study demonstrates that metabolomic profiling yields consistent results with LC50 values, and is valuable to elucidate molecular-level perturbations in soil organisms.