Glutathione transferases (GSTs) are enzymes that catalyse modifications and conjugations of a range of organic and often cytotoxic compounds. GST enzymes with many functions—such as their conjugation activity against herbicides and their metabolites—can be induced and show light and circadian determination. The enzyme family, which is widespread in its function, also shows great diversity in its structure, which has been linked to its enzyme kinetic characteristics and physiological role at many points. In this study, we aimed to find out the role of different glutathione transferases in the herbicide responses to flumioxazin, as well as to determine how the antioxidant and detoxification response to herbicide treatment changes in the presence and absence of light. One of the herbicide treatments was carried out during the light period in the morning (9:00 a.m.), and the other before the end of the dark period (4:00 a.m.). The decrease in the maximal quantum efficiency of PS II and the reduction in the chlorophyll concentration supported the effect of the herbicide on Papaver rhoeas. In the guaiacol peroxidase POD and GST activity, there were large differences between the cultivated plants and the weed; both enzyme activities were much higher in the case of wheat. According to the activity of the antioxidant defence enzymes and GST gene expression data, the application of the photosynthesis inhibitor herbicide, flumioxazin, in the dark could allow the wheat antioxidant defence to switch on before the herbicide effect could appear in the light period. Phi and tau group GSTs were transcriptionally upregulated by the treatments in wheat plants (especially TaGSTU1B), while fewer changes were detectable in poppy weed (PrGSTU4). Based on our results, in the background of the greater and more successful response to flumioxazin may be—among other things—the higher degree of variability of the GSTU genes of wheat compared to poppies.