Cellular senescence of normal human cells has by now far exceeded its initial role as a model system for aging research. Many reports show the accumulation of senescent cells in vivo, their effect on their microenvironment and its double-edged role as tumour suppressor and promoter. Importantly, removal of senescent cells delays the onset of age-associated diseases in mouse model systems. To characterize the role of miRNAs in cellular senescence of endothelial cells, we performed miRNA arrays from HUVECs of five different donors. Twelve miRNAs, comprising hsa-miR-23a, hsa-miR-23b, hsa-miR-24, hsa-miR-27a, hsa-miR-29a, hsa-miR-31, hsa-miR-100, hsa-miR-193a, hsa-miR-221, hsa-miR-222 and hsa-let-7i are consistently up-regulated in replicatively senescent cells. Surprisingly, also miR-21 was found up-regulated by replicative and stress-induced senescence, despite being described as oncogenic. Transfection of early passage endothelial cells with miR-21 resulted in lower angiogenesis, and less cell proliferation mirrored by up-regulation of p21CIP1 and down-regulation of CDK2. These two cell-cycle regulators are indirectly regulated by miR-21 via its validated direct targets NFIB (Nuclear factor 1 B-type), a transcriptional inhibitor of p21CIP1, and CDC25A, which regulates CDK2 activity by dephosphorylation. Knock-down of either NFIB or CDC25A shows a phenocopy of over-expressing miR-21 in regard to cell-cycle arrest. Finally, miR-21 over-epxression reduces the replicative lifespan, while stable knock-down by sponges extends the replicative lifespan of endothelial cells. Therefore, we propose that miR-21 is the first miRNA that upon its knock-down extends the replicative lifespan of normal human cells.