The proliferative lifespan of normal somatic human cells in culture terminates in a permanent growth-arrested state known as replicative senescence. In this study, we show that RNA interference-mediated repression of the genes encoding the small ubiquitin-related modifier (SUMO)-specific proteases, Senp1, Senp2, and Senp7, induced low passage primary human fibroblasts to senesce rapidly. Following Senp1 repression, we observed a global increase in sumoylated proteins and in the number and size of nuclear SUMO-containing promyelocytic leukemia (PML) bodies. SUMO/PML bodies also increased during replicative senescence. p53 transcriptional activity was enhanced towards known p53 target genes following repression of Senp1, and inhibition of p53 function prevented senescence after Senp1 repression. These data indicate that Senp1 repression induces p53-mediated premature senescence and that SUMO proteases may thus be required for proliferation of normal human cells.