6PPD-quinone (6PPD-Q) has been identified as a ubiquitous contaminant in the surrounding locality, including air particles, roadside soils, dust, and water. Recently, the prevalence of 6PPD-Q in human urine has accentuated the urgency for investigating its biological fate. To address this, we conducted a stable isotope-assisted high-resolution mass spectrometry (HRMS) assay to unveil the distribution, metabolism, excretion, and toxicokinetic properties of this contaminant in a mouse model. Mice were fed with a single dose of deuterated 6PPD-Q-d5 at human-relevant exposure levels. Results indicated that 6PPD-Q was quickly assimilated and distributed into bloodstream and main organs of mice, with the concentrations reaching peaks under 1 h following administration. Notably, 6PPD-Q was primarily distributed in the adipose tissue, marked by a significant Cmax (p < 0.05), followed by the kidney, lung, testis, liver, spleen, heart, and muscle. In addition, our measurement demonstrated that 6PPD-Q can penetrate the blood-brain barrier of mice within 0.5 h after exposure. The half-lives (t1/2) of 6PPD-Q in serum, lung, kidney, and spleen of mice were measured at 12.7 ± 0.3 h, 20.7 ± 1.4 h, 21.6 ± 5.3 h, and 20.6 ± 2.8 h, respectively. Using HRMS combined with isotope tracing techniques, two novel hydroxylated metabolites of 6PPD-Q in the mice liver were identified for the first time, which provides new insights into its rapid elimination in-vivo. Meanwhile, fecal excretion was identified as the main excretory pathway for 6PPD-Q and its hydroxylated metabolites. Collectively, our findings extend the current knowledge on the biological fate and exposure status of 6PPD-Q in a mouse model, which has the potential to be extrapolated to humans.