Chronic Mg2+ deficiency is at the origin of a broad range of health dysfunctions. Because 25% of body Mg2+ is located in the skeletal muscle, Mg2+ transport and homeostasis systems (MgTHs) in muscle are critical for whole body Mg2+ homeostasis. In the present study, we assessed whether Mg2+ deficiency alters muscle fiber characteristics and major pathways regulating muscle physiology. C57BL/6J mice were fed for 14 days either a control, mildly or severely Mg2+-deficient diet (0.1%; 0.01%; and 0.003% Mg2+ wt/wt, respectively). Mg2+ deficiency slightly decreased body weight gain, muscle Mg2+ concentrations, but was not associated with detectable variations in gastrocnemius muscle weight, fiber morphometry and capillarization. Nonetheless, to maintain Mg2+ homeostasis muscles exhibited decreased expression of several MgTHs (Magt1, Cnnm2, Cnnm4 and Trpm6). MgTHs mRNA levels were positively correlated with body weight gain. Moreover, TaqMan Low Density Array (TLDA) analyses further revealed that, before the emergence of major muscle dysfunctions, even a mild Mg2+ deficiency was sufficient to alter expression of genes critical for muscle physiology, including energy metabolism, muscle regeneration, proteostasis, mitochondrial dynamics and excitation-contraction coupling.