Cardiac glycosides (CGs), potent inhibitors of Na⁺,K⁺-ATPase, have been used to treat congestive heart failure. Recently, the effectiveness of CGs for cancer therapy has been suggested. Here, we examined the effects of CGs on glucose metabolism in human cancer cells. Low concentrations (nM levels) of CGs (ouabain, oleandrin, and digoxin) significantly decreased the expression level of glucose transporter GLUT1 in the plasma membrane of the cancer cells. Ouabain (20-2000 nM) inhibited 2-deoxy-D-glucose uptake and lactate secretion of cancer cells. In intracellular vesicles of human cancer cells, Na⁺,K⁺-ATPase α3-isoform (α3NaK) is abnormally expressed. Interestingly, the knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression, while the α1NaK knockdown did not. Ouabain (200 nM) inhibited the enzyme activity of α3NaK but not α1NaK. The ouabain-induced GLUT1 decrease was significantly inhibited by a Ca²⁺ chelator, a Ca²⁺-ATPase inhibitor, an NAADP antagonist, a dynamin inhibitor, and PI3K inhibitors. These results suggest that CGs act on intracellular α3NaK and induce the NAADP-mediated Ca²⁺ mobilization and PI3K activation followed by dynamin-dependent GLUT1 endocytosis. This mechanism may explain why CGs inhibit glucose uptake and glycolysis in human cancer cells.