Normal cell growth can be permanently blocked when cells enter a state known as senescence. This phenomenon can be triggered by various stresses, such as replicative exhaustion, oncogenic stimulation, or oxidative stress. Senescence prevents transmission of aberrant signals to daughter cells and thus prevents irreversible damage that could favor cancer development. To identify new genetic events controlling senescence, we have performed a loss-of-function genetic screen on normal human cells. We report that knockdown of topoisomerase I (Top1) results in an increased replicative potential associated with a decrease in senescence markers and a diminished DNA damage response. In addition, Top1 depletion also favors a bypass of oncogene-induced senescence. Conversely, Top1 constitutive expression induces growth arrest, the appearance of a senescence marker, and an activation of the DNA damage response. Altogether, these results reveal an unanticipated function of Top1 in regulating senescence. [Cancer Res 2009;69(10):4101–6]