Differentiation-inducing factor 1 (DIF-1) is a polyketide produced by slime mold Dictyostelium discoidium which inhibits growth and migration and promotes differentiation of Dictyostelium cells by localizing to mitochondria. DIF-1 regulates phosphorylation of signaling molecules involved in rearrangement of actin cytoskeleton and induces transient cortical accumulation of F-actin in Dictyostelium cells. We recently reported that DIF-1 inhibits growth and migration of various types of mammalian cell lines through, at least in part, activation of AMP-activated kinase (AMPK). However, the molecular mechanism for the effect of DIF-1 on actin dynamics remains elusive. Here, we found that DIF-1 regulates actin cytoskeleton in endothelial cells by activating Cofilin, an actin depolymerization factor. In mouse immortalized endothelial cells (SVECs), DIF-1 rapidly induced dephosphorylation and activation of cofilin, followed by actin fiber depolymerization. When Cofilin is activated, mitochondrial fission is induced. Consistently, DIF-1 induced mitochondrial fission and knockdown of Cofilin suppressed mitochondrial fission by DIF-1. These results suggested that DIF-1 regulates actin cytoskeleton and induces mitochondrial fission via Cofilin activation.