Steep redox gradients and diverse microbial communities in the anaerobic hyporheic zone create complex pathways for the degradation of herbicides, often linked to various terminal electron-accepting processes (TEAPs). Identifying the degradation pathways and their controlling factors under various TEAPs is of great significance for understanding mechanisms of water purification in the hyporheic zone. However, current research on herbicides in this area remains insufficient. Acetochlor, a commonly detected herbicide in aquatic environments, was the target contaminant in this study. Biogeochemical data, transformation products examination, and compound-specific isotope analysis (CSIA) were used to elucidate the degradation mechanisms of acetochlor under various TEAPs in anaerobic microcosms with hyporheic sediments. Results showed that carbon isotope fractionation of acetochlor during abiotic reduction by reduced sulfur species (ε