The human cytomegalovirus (HCMV) induces a replicative senescence program after arresting host cell cycle progression so as to create a favorable environment for its replication. Here, we report that HCMV infection stimulates the expression of p16(INK4a), a direct effector of the senescence phenotype. The increase in p16(INK4a) gene expression was due to an increase in gene transcription, since the expression of a reporter gene driven by the p16(INK4a)-encoding CDKN2A gene promoter was strongly induced by HCMV infection. The results of deletion and mutational analysis of the CDKN2A promoter further suggest the involvement of Ets transcription factors in HCMV-mediated stimulation of p16(INK4a) gene expression. The significance of p16(INK4a) upregulation during the HCMV replicative cycle is underscored by the finding that virus replication was severely impaired in fibroblasts homozygous for an intragenic deletion in CDKN2A locus and devoid of functional p16(INK4a). Moreover, a retrovirus-mediated p16(INK4a) small interfering RNA (p16-siRNA) effectively reduced viral replication, thus providing direct evidence that p16(INK4a) upregulation plays a positive role for HCMV replication.