Decreased potential of spermatozoa fertility is closely associated with the development of oxidative stress and dysfunction of ion-transporting ATPases. Oxidative stress may have negative impact on the activity of membrane-bound enzymes, such as Са 2+ ,Мg 2+ -АТPase that is involved in maintaining calcium homeostasis in sperm cells. The aim of present work was to evaluate the exogenous H 2 O 2 effect on the main kinetic parameters of ATP hydrolysis by plasma membrane Са 2+ ,Мg 2+ -АТPase of spermatozoa of fertile (normozoospermia) and infertility (asthenozoospermia) men. Since Са 2+ ,Mg 2+ -АТPase is one of the targets for the reactive oxygen species and is directly involved in oxidative stress, spermatozoa obtained from normo- and asthenozoospermic samples were subjected to oxidative stress in the form of exogenous H 2 O 2 . Then ATP hydrolysis by thapsigargin-resistant Ca 2+ ,Mg 2+ -ATPase in media with different Ca 2+ concentrations was measured. An effective inhibitory effect of H 2 O 2 on the activity of the thapsigargin-resistant component of Са 2+ ,Мg 2+ -АТPase of sperm cells was demonstrated. In order to elucidate possible mechanisms of change in Ca 2+ ,Mg 2+ -ATPase activity under H 2 O 2 -induced oxidative stress, the concentration curves were linearized using Hanes–Woolf plot {[S]/V; [S]}. The apparent activation constant for Ca 2+ (K Ca2+ ) in sperm cell obtained from men with proven fertility was not changed, whereas in the asthenozoospermic samples, it was decreased almost twice under H 2 O 2 -induced oxidative stress. These results indicate that in normozoospermic samples H 2 O 2 implements its inhibitory action through the mechanism of uncompetitive inhibition of plasma membrane Ca 2+ ,Mg 2+ -ATPase activity. According to formal features of kinetics in the asthenozoospermic samples a mixed type of enzyme inhibition occurs under the oxidative stress induced by H 2 O 2 . Strategies to protect against a loss in Cа 2+ ,Mg 2+ -ATPase activity may be useful to prevent the harmful biochemical cascades leading to Ca 2+ overload and dysfunction of spermatozoa as a result of the oxidative stress.