Although the migration of hepatic stellate cells (HSCs) is important for hepatic fibrosis, the regulation of HSC migration is poorly understood. Interestingly, transforming growth factor (TGF)-β1 induces monocyte migration to sites of injury or inflammation in the early phase but inhibits cell migration in the late phase. In this study, we investigated the role of RhoA signaling in TGF-β1-induced HSC migration. We found that TGF-β1 increased the protein and mRNA levels of α-SMA and collagen type I in HSC-T6 cells. The level of RhoA-GTP in TGF-β1-stimulated cells was significantly higher than that in control cells. Moreover, cofilin phosphorylation and F-actin formation was more strongly detected in TGF-β1-stimulated cells than in control cells. Additionally, TGF-β1 induced the activation of NF-κB and the expression of extracellular matrix proteins and several cytokines in HSC-T6 cells. The active form of Rap1 (Rap1 V12) suppressed RhoA-GTP levels, whereas the dominant negative form of Rap1 (Rap1 N17) augmented RhoA-GTP levels. Therefore, we confirmed that Rap1 regulates RhoA activation in TGF-β1-stimulated HSC-T6 cells. These findings suggest that TGF-β1 regulates Rap1, resulting in RhoA suppression, NF-κB activation and F-actin formation during the migration of HSCs.