Body temperature is maintained in a narrow range in mammals, primarily controlled by sweating. In humans, the dynamic thermoregulatory organ, comprised of 2–4 million sweat glands distributed over the body, can secrete up to 4 L of sweat per day, thereby making it possible to withstand high temperatures and endure prolonged physical stress (e.g., long-distance running). The genetic basis for sweat gland function, however, is largely unknown. We find that the forkhead transcription factor, FoxA1, is required to generate mouse sweating capacity. Despite continued sweat gland morphogenesis, ablation of FoxA1 in mice results in absolute anihidrosis (lack of sweating). This inability to sweat is accompanied by down-regulation of the Na-K-Cl cotransporter 1 (Nkcc1) and the Ca²⁺-activated anion channel Bestrophin 2 (Best2), as well as glycoprotein accumulation in gland lumens and ducts. Furthermore, Best2-deficient mice display comparable anhidrosis and glycoprotein accumulation. These findings link earlier observations that both sodium/potassium/chloride exchange and Ca²⁺ are required for sweat production. FoxA1 is inferred to regulate two corresponding features of sweat secretion. One feature, via Best2, catalyzes a bicarbonate gradient that could help to drive calcium-associated ionic transport; the other, requiring Nkcc1, facilitates monovalent ion exchange into sweat. These mechanistic components can be pharmaceutical targets to defend against hyperthermia and alleviate defective thermoregulation in the elderly, and may provide a model relevant to more complex secretory processes.