N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q), an oxidative derivative of tire anti-degradant, has been linked to mortality in coho salmon (Oncorhynchus kisutch) and has exhibited potential human toxicity. Hence, exploring how 6PPD-Q interacts with biomacromolecules like enzymes is indispensable to assess its human toxicity and elucidate its mechanism of action. This investigation aims to explore the impact of 6PPD-Q on lactate dehydrogenase (LDH) through various methods. The findings indicate that 6PPD-Q can spontaneously embed in the coenzyme site of LDH and obviously change the biological activity of LDH by non-competitive inhibition. Simultaneously, this inhibitory effect is concentration-dependent. 6PPD-Q can affect both the level of LDH and the transcription of Ldha in AML-12 cells. Hydrogen bonding and van der Waals forces serve as the primary driving forces in LDH-6PPD-Q combination process. The apparent binding constant (K