Differentiation-inducing factor-1 (DIF-1), a morphogen produced by the cellular slime mold Dictyostelium discoideum, is a natural product that has attracted considerable attention for its antitumor properties. Here, we report a novel inhibitory effect of DIF-1 on the activation of hepatic stellate cells (HSCs) responsible for liver fibrosis. DIF-1 drastically inhibited transdifferentiation of quiescent HSCs into myofibroblastic activated HSCs in a concentration-dependent manner, thus conferring an antifibrotic effect against in the liver. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, showed any effect on the inhibition of HSC activation by DIF-1. In contrast, TWS119, a glycogen synthase kinase 3β (GSK3β) inhibitor, attenuated the inhibitory effect of DIF-1. Moreover, the level of inactive GSK3β (phosphorylated at Ser9) was significantly reduced by DIF-1. DIF-1 also inhibited nuclear translocation of β-catenin and reduced the level of non-phospho (active) β-catenin. These results suggest that DIF-1 inhibits HSC activation by disrupting the Wnt/β-catenin signaling pathway through dephosphorylation of GSK3β. We propose that DIF-1 is a possible candidate as a therapeutic agent for preventing liver fibrosis. •A cellular slime mold product DIF-1 blocked hepatic stellate cell (HSC) activation.•An inhibitor of glycogen synthase kinase (GSK) 3β attenuated the effect of DIF-1.•The level of inactive GSK3β was significantly reduced by DIF-1.•DIF-1 reduced nuclear translocation of β-catenin and the level of active β-catenin.•DIF-1 is a possible candidate for liver fibrosis therapy to target HSC activation.