Digoxin, an inhibitor of Na+/K+ ATPase, has been used in the treatment of heart-related diseases (such as congestive heart failure and atrial arrhythmia) for decades. Recently, it was reported that digoxin is also an effective HIF-1 alpha inhibitor. We investigated whether digoxin could suppress tumor cell growth through HIF-1 alpha in non-small cell lung cancer cells (A549 cells) under hypoxic conditions. An MTT assay was used to measure cell viability. RT-PCR and western blotting were performed to analyze the mRNA and protein expression of VEGF, NDRG1, and HIF-1 alpha. HIF-1 alpha nuclear translocation was then determined by EMSA. Digoxin was found to inhibit the proliferation of A549 cells under hypoxic conditions. Our results showed that hypoxia led to the upregulation of VEGF, NDRG1, and HIF-1 alpha both at the mRNA and protein levels. We also found that the hypoxia-induced overexpression of VEGF, NDRG1, and HIF-1 alpha was suppressed by digoxin in a concentration-dependent manner. As expected, our EMSA results demonstrated that under hypoxic conditions HIF-1 alpha nuclear translocation was also markedly reduced by digoxin in a concentration-dependent manner. Our results suggest that digoxin down-regulated hypoxia-induced overexpression of VEGF and NDRG1 at the transcriptional level probably through the inhibition of HIF-1 alpha synthesis in A549 cells.