Cordycepin (3′-deoxyadenosine) is a major bioactive agent in Cordyceps militaris, a fungus used in traditional Chinese medicine. It has been proposed to have many beneficial metabolic effects by activating AMP-activated protein kinase (AMPK), but the mechanism of activation remained uncertain. We report that cordycepin enters cells via adenosine transporters and is converted by cellular metabolism into mono-, di-, and triphosphates, which at high cordycepin concentrations can almost replace cellular adenine nucleotides. AMPK activation by cordycepin in intact cells correlates with the content of cordycepin monophosphate and not other cordycepin or adenine nucleotides. Genetic knockout of AMPK sensitizes cells to the cytotoxic effects of cordycepin. In cell-free assays, cordycepin monophosphate mimics all three effects of AMP on AMPK, while activation in cells is blocked by a γ-subunit mutation that prevents activation by AMP. Thus, cordycepin is a pro-drug that activates AMPK by being converted by cellular metabolism into the AMP analog cordycepin monophosphate. Display omitted •Cordycepin (100 μM) activates AMPK in human cells; higher concentrations are toxic•Cordycepin is taken up into cells and converted into mono-, di-, and triphosphates•AMPK activation correlates with the cellular content of cordycepin monophosphate•Cordycepin monophosphate mimics all three effects of AMP on AMPK in cell-free assays Cordycepin is a major bioactive component of Cordyceps militaris, a parasitic fungus much prized in traditional Chinese medicine. Hawley et al. report that it is taken into cells and converted by the metabolism into mono-, di-, and triphosphates. Cordycepin monophosphate activates AMPK by mimicking multiple effects of its natural activator, AMP.