Magnesium ions play an important role in cell biology. Cellular magnesium homeostasis responds to the same basic tenets in both prokaryotes and eukaryotes. Total cellular Mg2+ content is maintained below the concentration predicted by its electrochemical equilibrium and cellular free Mg2+ content is slightly below the concentration present in the extracellular environment. A similar modest gradient is present between the cytoplasm and the mitochondrial matrix or the lumen of the endoplasmic reticulum. In plant cells and eukaryotes, the distribution of Mg2+ within the cytoplasm and cellular organelles is well defined and Mg2+ homeostasis is tightly regulated through a combination of transport and chelating mechanisms. All organisms require adequate cellular Mg2+ levels to regulate the activity of numerous enzymes, channels and genes that directly and indirectly control metabolic and bioenergetics processes. Dynamic changes in total and free Mg2+ concentrations have been observed in both prokaryotes and eukaryotes following environmental, metabolic and hormonal variations. Although the changes in free Mg2+ concentrations are small in percentage terms compared to those of Ca2+, they are still of an amplitude sufficient to modulate the activity of the various cellular enzymes and processes. At the molecular level, our knowledge of Mg2+ transporters is more advanced in prokaryotes than in eukaryotes, although several of the transporters identified in prokaryotes have orthologues in both plant and mammalian cells. In eukaryotes, ATP and cAMP are essential chelating and regulating agents for Mg2+. Cyclic-AMP, especially, plays a key role in coordinating the effect of Mg2+ on cell metabolism and bioenergetics. Taken together, these pieces of evidence suggest an essential, albeit often underappreciated, role for Mg2+ as a key regulator of metabolic and cellular processes. Mounting clinical and experimental evidence further underlies the importance of this regulatory role in certain pathological conditions and diseases in humans, animals and plants alike.