The ATPase activity of myosin obligatory depends upon availability of Mg2+ ion that is necessary for binding of ATP in the active center of the enzyme together with the essential role of Mg2+ in the enzyme hydrolysis of ATP. Among three stable isotopes of magnesium, 24Mg, 25Mg and 26Mg with natural abundance about 79, 10 and 11%, only 25Mg has the nuclear spin (I = 5/2) and, hence, the nuclear magnetic moment. Two other isotopes, 24Mg and 26Mg, have no nuclear spin (I = 0) and no magnetic moment. We studied effects of the different isotopes of magnesium on ATP hydrolysis driven by the catalytic fragment (subfragment-1) of myosin isolated from myometrium. The enzyme activity has turned out to be 2 – 2.5 times higher in the reaction media enriched with 25Mg as compared to the activity of the same enzyme in the reaction media enriched with the spinless isotopes, 24Mg or 26Mg, or the enzyme activity in the media with natural Mg (natural isotope abundance). The catalytic effect of the nuclear spin of 25Mg was observed at physiological concentration, 5 mM, of 25MgCl2. Thus, we have documented, for the first time, the magnetic isotope effect (MIE) in the enzymatic ATP hydrolysis driven by myosin. At that, no magnetic isotope effect has been detected in the non-enzymatic, spontaneous, ATP hydrolysis. Factual evidence of MIE, on its own, indicates a “bottle-neck” that undergoes the spin conversion, i.e., that a spin-selective intermediate such as a free-radical pair, ion-radical pair, or conformational excitation of the macromolecule limits the rate of the reaction. Obviously, the nuclear spin of 25Mg accelerates the chemo-mechanical cycle of myosin, thus setting the enzyme macromolecule for acceptance and hydrolysis of next ATP molecules. The detailed physicochemical mechanisms of the nuclear spin catalysis require further investigations. [Supported by RFBR, project no. 14-04-00593a].