Polyamine levels are elevated in the organs and tissues of cancer patients due to increased synthesis and active intercellular transport in cancer cells. Because increased polyamine levels are associated with poor prognosis, the effect of polyamines on the malignant potential of cancer cells was investigated. Highly metastatic colon cancer cells (HT-29) were cultured under either normoxia (21% O2) or hypoxia (2% O2) for 48 h with 0, 100, or 500 µM spermine, one of the natural polyamines with the strongest biological activity. Spermine supplementation ameliorated MTT metabolism of hypoxic cancer cells in a dose-dependent manner, but had no effect on cells cultured under normoxia. Hypoxia decreased cancer cell CD44 and E-cadherin expression, while CD44 expression further decreased by spermine in a dose-dependent manner. By comparing cells cultured under normoxia with increasing amounts of spermine, we found that CD44 expression decreased by 11% (0 µM spermine), 14% (100 µM), and 18% (500 µM), and was accompanied by comparable decreases in CD44 mRNA levels. Martigel invasion assay showed that hypoxia increased the number of invading cells, and spermine further enhanced the hypoxia-induced increase in the number of invading cells in a dose-dependent manner. The numbers of invading cells cultured with 0, 100, and 500 µM spermine under hypoxia were 2.3, 2.8, and 3.2 times greater, respectively, compared to cells with 0 µM spermine under normoxia. Increased extracellular spermine enhances the invasion potential of cancer cells under hypoxia.