Acetochlor, a commonly used herbicide, poses significant risks to ecosystem and organism health through contamination of the food chain. Despite its widespread use, there is a lack of comprehensive studies on its toxicological effects on avian species. This study investigates the impact of environmental acetochlor exposure on chicken liver health using metabolomics analysis and histopathological techniques. Microscopic examination revealed autophagy-like structures and endoplasmic reticulum (ER) expansion, with significant effects observed at higher exposure levels. Biochemical analysis and metabolomics also demonstrated acetochlor-induced ferroptosis, highlighting disruptions in liver function. Further, in vitro studies revealed that acetochlor stimulates autophagy, which regulates ferroptosis via ferritin degradation, mediated through the ER-CaMKII pathway. These findings emphasize the importance of understanding the molecular mechanisms involved in acetochlor toxicity, particularly the role of the Ca