The presence of antibiotics in municipal wastewater is bound to affect the anoxic denitrifying process in anoxic activated sludge (AAS). This study investigated the effects of sulfamethoxazole (SMZ) on the denitrifying process in AAS and the responses of denitrifying microorganisms. The results showed that SMZ could decrease the speed of nitrate removal significantly when the concentration of SMZ was lower than 10 mg/L, and the removal of nitrate would be completely inhibited when SMZ concentration was higher than 100 mg/L. Weak alkaline condition would enhance the inhibition effect of SMZ on removal of nitrate in anoxic bioreactor. The results of high-throughput sequencing and qPCR showed that 100 mg/L of SMZ did not decrease the total abundance of denitrifying microorganisms. However, the relative expression levels of key denitrifying genes NirS and NosZ in AAS treated by 100 mg/L of SMZ versus the raw AAS without SMZ was only 0.030 and 0.036. Therefore, the inhibitory mechanism of SMZ on the denitrifying process in AAS was denoted by an effective inhibition to the expressions of denitrifying genes, rather than a decrease in the total abundance of denitrifying microorganisms.