Differentiation-inducing factor 1 (DIF-1) is a polyketide produced by slime mold Dictyostelium discoidiumwhich inhibits growth and migration and promotes differentiation of Dictyostelium cells by unknown mechanism. In addition to Dictyostelium, DIF-1 also inhibits growth and migration of various types of mammalian cells, at least in part, by activating AMP-activated protein kinase (AMPK). We previously reported that, in mammalian cells, DIF-1 binds to mitochondrial malate dehydrogenase (MDH2) and inhibits its enzymatic activity, although the physiological meaning of the binding has remained elusive. Here we report that inhibition of MDH2 by DIF-1 induces phosphorylation and activation of AMPK. Like DIF-1, LW6, a synthetic inhibitor for MDH2, regulated phosphorylation of AMPK and its downstream molecules such as p70S6 kinase and Cofilin. Knockdown of MDH2 by siRNA also induced phosphorylation of AMPK. In contrast, disease-associated mutants of MDH2 with high enzymatic activity inhibited phosphorylation of AMPK by DIF-1. In vitro, MDH2 activity was inhibited by both DIF-1 and its analogue, DIF-3. Taken together, we propose that a mammalian target of DIF-1 is mitochondrial malate dehydrogenase.