Overproduction of lactate (LA) can occur during exercise and in many diseases such as cancers. Individuals with hyperlactatemia often display anemia, decreased serum iron, and elevated hepcidin, a key regulator of iron metabolism. However, it is unknown whether and how LA regulates hepcidin expression. Here, we show LA binds to soluble adenylyl cyclase (sAC) in normal hepatocytes and affects systemic iron homeostasis in mice by increasing hepcidin expression. Comprehensive in vitro, in vivo, and in silico experiments show that the LA-sAC interaction raises cyclic adenosine monophosphate (cAMP) levels, which activates the PKA-Smad1/5/8 signaling pathway to increase hepcidin transcription. We verified this regulatory axis in wild-type mice and in mice with disordered iron homeostasis. LA also regulates hepcidin in humans at rest and subjected to extensive exercise that produce elevated LA. Our study links hyperlactatemia to iron deficiency, offering a mechanistic explanation for anemias seen in athletes and patients with lactic acidosis.