The transient receptor potential vanilloid 3 channel (TRPV3) is abundantly expressed in epidermal keratinocytes and has important roles in sensory biology and skin health. Mg2+ deficiency causes skin disorders under certain pathological conditions such as type 2 diabetes mellitus. In this study, we investigated the effect of Mg 2 on TRPV3 in primary epidermal keratinocytes. Extracellular Mg2+ (Mg2+]o) inhibited TRPV3-mediated membrane current and calcium influx. TRPV3 activation induced a calcium signaling pathway culminating in activation of the cAMP response element binding. TRPV3 inhibition by [Mg2+]o, the TRPV3 blocker ruthenium red, or TRPV3 siRNA suppressed this response. In TRPV3-expressing Chinese hamster ovary cells, both extracellular and intracellular Mg 2+ inhibited TRPV3 single-channel conductance, but not open probability. Neutralization of an aspartic acid residue (D641) in the extracellular pore loop or two acidic residues (E679, E682) in the inner pore region significantly attenuated the inhibitory effect of extracellular or intracellular Mg2+ on TRPV3-mediated signaling, respectively. Our findings suggest that epidermal TRPV3 is tonically inhibited by both extracellular and intracellular Mg2+, which act on both sides of the channel pore loop. Mg2+ deficiency may promote the function of TRPV3 and contribute to the pathogenesis of skin diseases.