The aim of this work was to investigate changes in isoenzyme patterns of enzymes related to reactive oxygen species (ROS) detoxification such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), and glutathione-S-transferase (GST) in cotton under Mg deficiency. Moreover, we aimed to elucidate how a ROS producer, NADPH oxidase (NOX), responds to changing Mg levels. Cotton plants were grown with different concentrations of MgSO4 in hydroponic medium to create nutrient deficiency (0, 75, 150, 1000 μM Mg). Gradual decreases in growth and photosynthetic rates were observed with declining Mg concentrations and 0, 75, and 150 μM Mg increased oxidative stress as evidenced by H2O2 and lipid peroxidation. Total activities of SOD, CAT, POX, APX, GR, and GST were increased while NOX activity was decreased with Mg deficiency. The activities of GR and GST were highest in plants treated with 0 μM Mg, indicating excess use of glutathione for redox regulation. The most striking results were the changes in isoenzyme patterns of SOD, NOX, POX, and GST. For example, a new Cu/ZnSOD isoenzyme was induced in plants treated with 0 μM Mg. Cotton plants adapt to Mg deficiency by changing the intensity of existing isoenzymes or inducing new ones.