The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier to tumour progression in vivo^,,. We show that the human p53 isoforms Δ133p53 and p53β function in an endogenous regulatory mechanism for p53-mediated replicative senescence. Induced p53β and diminished Δ133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expressed increased levels of miR-34a, a p53-induced microRNA^,,,,, the antisense inhibition of which delayed the onset of replicative senescence. The siRNA (short interfering RNA)-mediated knockdown of endogenous Δ133p53 induced cellular senescence, which was attributed to the regulation of p21^WAF1 and other p53 transcriptional target genes. In overexpression experiments, whereas p53β cooperated with full-length p53 to accelerate cellular senescence, Δ133p53 repressed miR-34a expression and extended the cellular replicative lifespan, providing a functional connection of this microRNA to the p53 isoform-mediated regulation of senescence. The senescence-associated signature of p53 isoform expression (that is, elevated p53β and reduced Δ133p53) was observed in vivo in colon adenomas with senescent phenotypes^,. The increased Δ133p53 and decreased p53β isoform expression found in colon carcinoma may signal an escape from the senescence barrier during the progression from adenoma to carcinoma.