It is well established that cytochrome P450 monooxygenases (P450s) play a crucial role in herbicide metabolism and resistance evolution in weeds. In most documented cases, P450-mediated resistance is primarily conferred through the overexpression of P450 enzymes. However, the regulatory mechanisms underlying this overexpression remain poorly understood. In insects, amino acid substitutions that enhance P450-mediated metabolic detoxification have been clearly demonstrated as a key mechanism of insecticide resistance. In contrast, their potential role in herbicide resistance in weeds remains unclear. In this study, two CYP96A146 variants from flixweed [Descurainia sophia (L.) Webb ex Prantl], designated CYP96A146-S and CYP96A146-R, were heterologously expressed in Saccharomyces cerevisiae. These variants, which differ by four amino acid residues, were examined for their ability to metabolize model substrates and herbicides. The results indicated that both variants exhibited catalytic activity toward model substrates of p-nitroanisole, methoxyresorufin, ethoxyresorufin, 7-ethoxycoumarin, and benzo[a]pyrene, as well as toward the herbicides tribenuron-methyl, bensulfuron-methyl, and carfentrazone-ethyl. Notably, CYP96A146-R showed significantly higher catalytic activity than CYP96A146-S to both the model substrates (p-nitroanisole, methoxyresorufin, ethoxyresorufin, and 7-ethoxycoumarin) and the herbicides (tribenuron-methyl and carfentrazone-ethyl). These findings suggest that the amino acid substitutions are likely responsible for the enhanced metabolic capability of CYP96A146-R. Such mutations may induce conformational changes of CYP96A146 enzyme, facilitating more frequent molecular collisions between CYP96A146 and the substrates or herbicides, thereby improving catalytic efficiency.