Pyridine and its derivatives are widely used in many applications and inevitably cause extreme scenarios of serious soil contamination, which pose a threat to soil organisms. Still, the eco-toxicological effects and underlying mechanisms of pyridine-caused toxicity toward soil fauna have not been well established. Thus, earthworms (Eisenia fetida), coelomocytes, and oxidative stress-related proteins were selected as targeted receptors to probe the ecotoxicity mechanism of extreme pyridine soil exposure targeted to earthworms by using a combination of in vivo animal experiments, cell-based in vitro tests, in vitro functional and conformational analyses, and in silico analyses. The results showed that pyridine caused severe toxicity to E. fetida at extreme environmental concentrations. Exposure of pyridine induced excessive ROS formation in earthworms, causing oxidative stress and various deleterious effects, including lipid damage, DNA injury, histopathological change, and decreased defense capacity. Also, pyridine destroyed the cell membrane of earthworm coelomic cells and triggered a significant cytotoxicity. Importantly, the intracellular ROS (e.g., O, HO, and OH·) was release-activated, which eventually inducing oxidative stress effects (lipid peroxidation, inhibited defense capacity, and genotoxicity) through the ROS-mediated mitochondrial pathway. Moreover, the antioxidant defence mechanisms in coelomocytes responded quickly to reduce ROS-mediated oxidative injury. It was conformed that the abnormal expression of targeted genes associated with oxidative stress in coelomic cells was activated after pyridine exposure. Particularly, we found that the normal conformation (particle sizes, intrinsic fluorescence, and polypeptide backbone structure) of CAT/SOD was destroyed by the direct binding of pyridine. Furthermore, pyridine bound easily to the active center of CAT, but preferentially to the junction cavity of two subunits of SOD, which is considered to be a reason for impaired protein function in cells and in vitro. Based on these evidences, the ecotoxicity mechanisms of pyridine toward soil fauna are elucidated based on multi-level evaluation.