P53 homolog p63 was shown to play a role in premature ageing phenotype found in mouse models through regulation of the replicative senescence. We previously showed that the forced ΔNp63α expression decreased the SIRT1 protein levels, and induced the replicative senescence of human keratinocytes, while the ectopic SIRT1 expression decreased the senescence. Using the ΔNp63α overexpressing and p63-/+ heterozygous mice, we found that ΔNp63α induced the mTERT promoter activation through the down regulation of the SIRT1 protein levels, inactivation of p53 deacetylation, decrease of the p53/Sp1 protein-protein interaction, and the overall induction of mTERT transcription regulation. In the same time, by a forming of protein-protein complexes with the ABBP1, ΔNp63α induced the mTERT RNA splicing leading to an increasing expression of spliced mTERT isoforms playing a role of dominant-negative inhibitors of mTERT activity and therefore decreasing the levels of TERT activity in mouse epidermal keratinocytes. The overall effect of the ΔNp63α overexpression resulted in decrease in telomerase activity and increase in replicative senescence observed in mouse keratinocytes. This dual molecular mechanism of telomerase regulation might underline the previously shown effect of ΔNp63α on premature ageing phenotype.