Hepatic stellate cells (HSCs) are activated in response to liver injury and secrete huge amounts of collagen, the primary cause of liver fibrosis (LF). Thus, the regulation of trans-differentiation of HSCs, both activation of quiescent HSCs and reversion of activated HSCs, is crucial for therapeutic strategy for LF. However, few compounds have been reported to have such effects and no definitive therapy is available. Here, we elucidate the effect of DIF-1, a compound inhibiting HSC activation we previously reported, on activated HSC and LF mouse model. DIF-1 reduced the expression of type I collagen α 1 (Col1a1) and α-smooth muscle actin, markers of activated HSCs, even when treated after HSC activation. We further performed in silico analysis utilizing the relation between structural transition and HSC reversion effect of several DIF-1 analogs to identify molecular target of DIF-1. DIF-1 reduced the expression of activated HSC marker genes (Acta2, Col1a1, Pdgfrb), while it increased that of a quiescent HSC marker gene (Lrat) in thioacetamide-induced LF mouse model. Moreover, DIF-1 reduced the amount of collagen fiber in liver tissue. Taken together with our previous report, we propose that DIF-1 is a useful compound for LF treatment, acting on both quiescent and activated HSCs.