Digoxin (DG), digitoxin (DT), cardiac glycosides, and Na^+ -K^+ -ATPase inhibitors, are some of the traditional drugs used to treat heart disease. In a previous study, we administered DG and DT to Hepatocellular Carcinoma (HCC) cell lines and observed the resulting cell growth, viability, and migration. Our results showed that both DG and DT exhibit anticancer effects, such as decreasing cell proliferation and survival rate. In this study, cell migration and invasion were examined in transwell motility assays. We further observed F-actin polymerization and HIF-1α expression to identify cancer-related pathways. The results showed that DG treatment inhibited cell migration and invasion and also depolymerized F-actin. Finally, we found that DG treatment reduced HIF-1α expression in HCC cell lines. Our results indicated that the anticancer effects of DG on HCC cell lines include decreasing cell metastasis through decreasing cell viability, migration, invasion and F-actin depolymerization. Moreover, these protective functions might be associated with the suppression of the hypoxia response pathway. We believe that these novel findings opens a path toward new strategies in HCC treatment.