Hypoxia was shown to increase tumor cell invasion into the extracellular matrix in vitro. This result suggests that heparanase (Hpa), one of the key enzymes involved in tumor invasion and metastasis, may be regulated by hypoxia. RT-PCR, Western blot and Matrigel invasive assays were used to study the regulation of Hpa under hypoxia in human pancreatic MIA PaCa-2 cancer cells. Compared with those in normoxia (20% O2), Hpa mRNA, protein and enzymatic activity levels, were up-regulated by a reduction in the oxygen level (1% O2). Invasion by tumor cells into the extracellular matrix was found to be significantly enhanced. A reduction in Hpa protein levels was observed when nuclear factor kappaB (NF-kappaB) activation was blocked by pyrrolidine dithiocarbamate. The levels of Hpa were also reduced when Hpa was inhibited by an Hpa-specific antisense oligonucleotide. The MMP-9 mRNA, protein and gelatinase B activity levels in supernatants decreased significantly when Hpa was inhibited. We conclude that up-regulation of Hpa by hypoxia is NF-kappaB-dependent in MIA PaCa-2 cells and inhibition of Hpa reduces MMP-9 activity. This reduction in MMP-9 activity may be an important mechanism in tumor metastasis.