Hypoxia-inducible factor-1 (HIF-1) is a ubiquitously expressed oxygen-regulated transcription factor composed of α and β subunits. HIF-1 activates transcription of various genes including those involved in metastatic tumor growth. In the present study, HIF-1α expression in tumor-bearing mouse liver was examined after inoculation of tumor cells into portal vein. We found that tumor-bearing liver showed greatly increased HIF-1α expression. Plasmid DNA (pDNA) expressing short hairpin RNA targeting HIF-1α (pshHIF-1α) was effective in suppressing protein expression of HIF-1α in vitro. Intravenous injection of pshHIF-1α by hydrodynamics-based procedure reduced the HIF-1α protein expression in both normal and tumor cells and tumor cell number in the liver. Pre-injection of pshHIF-1α to mice, by which pDNA was delivered only to liver cells, not to tumor cells, was also effective in reducing the number of tumor cells inoculated 3 days after pDNA injection. These findings indicate that HIF-1α expression is increased in normal liver cells as well as tumor cells, and HIF-1α expression plays an important role in tumor progression. Use of the RNA interference (RNAi) of HIF-1 is an effective strategy for inhibiting tumor cell growth, and both tumor and normal cells can be the target for RNAi-based anticancer treatment.