Current Western diet is low in Mg2+ content. Prolonged exposure to Mg2+-deficient diet results in increased production of FAs and TGs, obesity, and ER stress. Because the ER is one of the major pools of cellular Mg2+, we investigated the effect that a decrease in ER Mg2+ content may have on the function of G6Pase enzymatic complex. Liver microsomes were purified from rats fed a Mg2+-deficient diet for two weeks. Our results indicate that the decrease in hepatic and reticular Mg2+ content stimulates G6P hydrolysis by ~40%. This effect could be reverted in vitro by the addition of 1mM extrareticular Mg2+, a dose that resembles cytosolic [Mg2+]i in the hepatocyte. The effect of Mg2+ was abolished by pre-treatment of microsomes with taurocholic acid, which bypasses the G6P entry mechanism into the microsome lumen. Microsomes isolated from livers acutely perfused with 1% EtOH for 10min showed a marked decrease in G6P hydrolysis. This effect occurred independent of the amount of Mg2+ present in the diet. Also in this case, G6P hydrolysis rate was restored by taurocholic acid pre-treatment, suggesting that EtOH affected the substrate binding site and not the catalytic site of the G6Pase complex. Conclusion These results indicate that the G6P binding site of G6Pase is dynamically modulated by cytoplasmic Mg2+ and EtOH metabolism, confirming its role as the limiting step of G6Pase hydrolytic activity.