Abstract The selenoprotein gastrointestinal glutathione peroxidase 2 (GPx2) is up-regulated in a variety of cancer cells with thus far unknown consequences. Therefore, two clones of a human colon cancer cell line (HT-29) in which GPx2 was stably knocked down by small interfering RNA (siRNA; siGPx2) were used to test whether cancer-relevant processes are affected by GPx2. The capacity to grow anchorage independently in soft agar was significantly reduced in siGPx2 cells when compared with controls (i.e., HT-29 cells stably transfected with a scramble siRNA). The weight of tumors derived from siGPx2 cells injected into nude mice was lower in 9 of 10 animals. In contrast, in a wound-healing assay, wound closure was around 50% in controls and 80% in siGPx2 cells, indicating an enhanced capacity of the knockdown cells to migrate. Similarly, invasion of siGPx2 cells in a Transwell assay was significantly increased. Migration and invasion of siGPx2 cells were inhibited by celecoxib, a cyclooxygenase-2 (COX-2)–specific inhibitor, but not by α-tocopherol. Selenium supplementation of cell culture medium did not influence the results obtained with siGPx2 cells, showing that none of the other selenoproteins could replace GPx2 regarding the described effects. The data show that GPx2 inhibits malignant characteristics of tumor cells, such as migration and invasion, obviously by counteracting COX-2 expression but is required for the growth of transformed intestinal cells and may, therefore, facilitate tumor cell growth. The data also shed new light on the use of selenium as a chemopreventive trace element: a beneficial effect may depend on the stage of tumor development. [Cancer Res 2008;68(23):9746–53]