Evening primrose (Oenothera paradoxa Hudziok) seeds are a rich source of not only a valuable oil containing an essential fatty acid - γ-linolenic acid (GLA) - but also polyphenols which can be obtained from the biomass remaining after oil pressing. The aim of our studies was to evaluate the influence of a polyphenol extract from defatted seeds of evening primrose on human colorectal adenocarcinoma Caco-2 cell proliferation and matrix metalloproteinases (MMPs) synthesis and activity. To assess the effect of evening primrose extract on the proliferation crystal violet staining and sulforhodamine B (SRB) assays were used whereas mRNA expression and activity of MMPs were evaluate by RT-PCR and gelatin zymography. The results revealed that the examined polyphenol extract has little influence on Caco-2 proliferation, but effectively in a time- and dose-dependent manner inhibits MMP-1, MMP-7, MMP-9 and MMP-14 mRNA synthesis induced by TNF-α and TPA. Additionally, zymographic analysis revealed that after 24 h, the polyphenol extract at a concentration of 50 μmol/L GAE caused a 10-fold reduction in MMP-9 synthesis. Moreover, this extract might be a potent inhibitor of MMP activity. The results showed that polyphenol extract from evening primrose inhibited PBMC-derived MMP-2 and MMP-9 enzymatic activity in dose-dependent manner. The obtained results indicate that the polyphenol extract from evening primrose seeds could be an inhibitor of proteases involved in tumor progression and metastasis.