The effects of sulfonamide antibiotics on heterotrophic nitrification-aerobic denitrification (HN-AD) and the response mechanisms of HN-AD bacteria are not fully understood. This study investigated the physiological changes and proteomic responses of the HN-AD bacteria Pseudomonas stutzeri (P. stutzeri) under varying concentrations of sulfamethoxazole (SMX). Results indicated that SMX inhibited the growth and HN-AD performance of P. stutzeri in a concentration-dependent manner. SMX exposure led to decreased motility, reduced electron transfer system activity, and diminished activities of key denitrifying enzymes, accompanied by increased levels of intracellular reactive oxygen species and compromised cell membrane integrity. Additionally, the production of extracellular polymeric substances and self-aggregation ability of P. stutzeri initially increased and then decreased with rising SMX concentrations. Proteomic analysis revealed that SMX primarily suppressed pathways involved in bacterial chemotaxis, ABC transporters, two-component systems, fatty acid metabolism, and nitrogen metabolism. In response, P. stutzeri upregulated pathways associated with starch and sucrose metabolism, carotenoid biosynthesis, styrene degradation, O-antigen nucleotide sugar biosynthesis, and the pentose phosphate pathway. These findings provide insights into the effects of sulfonamide antibiotics on HN-AD bacteria and their response mechanisms, offering references for the application of HN-AD processes in treating antibiotic-containing wastewater.