Riparian zones can receive large amounts of nitrate, potentially contributing to water pollution. Denitrification is a major pathway to remove nitrate. Previous research on riparian denitrification focused on natural factors, but frequently neglected the roles of human activity, such as pesticide accumulations. Here, we combined field investigations and exposure experiments to reveal the responses of denitrification and N2O emission to chlorothalonil (CTN, a common pesticide) in column experiments with riparian sediments. In this study, CTN inhibited denitrification and led to nitrate accumulation in sediments. Furthermore, CTN significantly increased N2O emission by 208–377%, and this response was regulated by N2O reductase (NOS) activity rather than nosZ abundance. A mechanistic study indicated that the critical step (glyceraldehyde-3-phosphate to 3-phosphogylcerate) catalyzed by glyceraldehyde-3-phosphate dehydrogenase during microbial metabolism greatly influenced denitrification in CTN-polluted sediments. Our data also revealed that CTN declined electron donor NADH, electron transport system, and denitrifying enzyme activities during denitrification. Such responses suggested that CTN deteriorated sediment denitrification by inhibiting electron production, transport and consumption in denitrifiers. Additionally, structure equation modeling indicated that NOS was the key factor in predicting denitrification rate in CTN-polluted sediments. Overall, this is the first study to explore the effects of pesticide on denitrification and N2O emission in riparian zones at microbial metabolism level. Our results suggest that the safety threshold of CTN accumulation for inhibiting sediment denitrification is approximately 2 mg kg−1, and imply that the wide presence of pesticides in riparian zones could impact eutrophication control of aquatic ecosystems. Display omitted •First study to test the effects of pesticides on denitrification in riparian sediments.•CTN significantly inhibited denitrification but stimulated N2O emission.•The critical step catalyzed by GAPDH in glycolysis greatly impacted denitrification.•N2O reductase was the key factor for predicting denitrification rate in CTN-polluted sediments.•Safety threshold of CTN for inhibiting denitrification should be no more than 2 mg kg−1.