The fate of antibiotics in aquatic environments is of high concern and approaches are needed to assess the transformation of antibiotics in wastewater treatment plants. Here we used the model organism Desulfovibrio vulgaris Hildenborough to analyze compound specific isotope fractionation associated with anaerobic transformation of the antibiotic sulfamethoxazole (SMX). The results show that the rearrangement of the isoxazole ring in SMX is leading to significant carbon and hydrogen isotopic fractionation (εC = −5.8 ± 0.7‰, εH = −34 ± 9‰) during anaerobic transformation. The observed carbon isotopic fractionation is significantly higher than the values reported for aerobic degradation (εC = −0.6 ± 0.1‰) or abiotic reactions (εC = −0.8 to −4.8‰ for photolysis, εC = −0.8 to −2.2‰ for advanced oxidation). This indicates that carbon isotope fractionation can be used as a parameter to differentiate reaction mechanisms of SMX transformation. The corresponding apparent kinetic isotope effect (AKIEC) for anaerobic transformation of SMX was 1.029 ± 0.003, suggesting that the mechanism for anaerobic transformation is distinct from the mechanism reported for microbial aerobic degradation (AKIEC = 1.006 ± 0.001). In addition, dual-element (C–H) isotope analysis of SMX was performed in the present study, which was achieved by utilizing gas chromatography (GC) as the separation method instead of routine liquid chromatography. This dual-element isotope analysis resulted in a Λ value of 4.5 ± 2.2. Overall, compound specific isotope analysis can be a feasible tool to monitor the mitigation of SMX in wastewater treatment plants.