Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the oxidative decarboxylation of isocitrate in the citric acid cycle. In the alpha(2)beta gamma heterotetramer of NAD-IDH, the gamma subunit plays the regulatory role and the beta subunit the structural role. Previous biochemical data have shown that mammalian NAD-IDHs can be inhibited by NADH; however, the molecular mechanism is unclear. In this work, we show that the alpha beta, alpha gamma and alpha(2)beta gamma enzymes of human NAD-IDH can be inhibited by NADH, and further determine the crystal structure of the alpha gamma heterodimer bound with an Mg2+ and an NADH at the active site and an NADH at the allosteric site, which resembles that of the inactive alpha(Mg)gamma heterodimer. The NADH at the active site occupies the binding site for NAD(+) and prevents the binding of the cofactor. The NADH at the allosteric site occupies the binding sites for ADP and citrate and blocks the binding of the activators. The biochemical data confirm that the NADH binding competes with the binding of NAD(+) and the binding of citrate and ADP, and the two effects together contribute to the NADH inhibition on the activity. These findings provide insights into the inhibitory mechanisms of the alpha gamma heterodimer by NADH.