Cardiac glycosides, which are inhibitors of Na⁺/K⁺-ATPase, are classified into cardenolides and bufadienolides. We have recently shown that two cardenolide glycosides, ouabain and odoroside A, inhibit Na⁺/K⁺-ATPase, thereby preventing nuclear factor κB-inducible protein expression by blocking Na⁺-dependent amino acid transport. In this study, we investigated the mechanism of action of cardenolide aglycones in tumor necrosis factor α (TNF-α)-induced gene expression. Ouabagenin, digitoxigenin, and digoxigenin were found to inhibit the TNF-α-induced cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) in human lung carcinoma A549 cells. Those cardenolide aglycones did not inhibit the TNF-α-induced expression of ICAM-1 mRNA, but strongly inhibited the TNF-α-induced expression of ICAM-1 as translation product. The inhibition of the TNF-α-induced ICAM-1 expression by ouabagenin, digitoxigenin, and digoxigenin was significantly reversed by the ectopic expression of ouabain-resistant rat Na⁺/K⁺-ATPase α1 isoform. Moreover, knockdown of Na⁺/K⁺-ATPase α1 isoform augmented the inhibition of the TNF-α-induced ICAM-1 expression by ouabagenin or ouabain. These results clearly indicate that cardenolide aglycones inhibit the TNF-α-induced ICAM-1 expression at the translation step by blocking Na⁺/K⁺-ATPase.