Vascular endothelial growth factor (VEGF) is necessary for angiogenesis and tumor growth. VEGF splice isoforms have distinct characteristics, with VEGF121 being more diffusible, thus allowing for more distant angiogenic signaling, and VEGF165 and VEGF189 being found within the cell and the extracellular matrix. We hypothesized that VEGF expression in cancer cells would increase under hypoxia and that the VEGF 121 isoform would be enhanced. We examined VEGF mRNA levels and relative isoform ratios in cells grown in hypoxia or treated with CoCl2 to mimic hypoxia. Prostate cancer (PC3, LNCaP) and leukemia (MOLT-4, K562) cells were initially cultured under normoxic conditions with 20% O2 and then placed in a hypoxia chamber with 1% O2 or treated with CoCl2. RNA was extracted from treated and control cells, and quantitative real-time PCR analysis was performed to analyze VEGF expression under the different conditions. Our findings suggest that hypoxia increased both VEGF121 and VEGF165 expression in prostate cancer and leukemia cells. These results of elevated VEGF121 indicate the potential for lethal metastatic tumor growth distant from the primary tumor site. Overall, this work highlights the role of the hypoxic tumor microenvironment in regulating the functionally distinct VEGF isoforms in cancer cells.