As a cyclin-dependent kinase inhibitor, p16(INK4a) plays a key role in cell cycle progression and cellular differentiation, and its expression is frequently altered in human cancers through epigenetically mediated transcriptional silencing. In this report, we demonstrate that p300 was able to induce cell cycle arrest, and this process was reversed by p16(INK4a) silencing by RNA interference in HeLa cells. We also show that p300 was involved in activation of p16(INK4a) expression in 293T cells. Specifically, p300 cooperated with Sp1 to stimulate both p16(INK4a) promoter activity and mRNA expression. Co-immunoprecipitation and mammalian two-hybrid assays revealed that p300 and Sp1 formed a complex through interaction between the Q domain of p300 and the N-terminal domain of Sp1. The chromatin immunoprecipitation assays verified that p300 was recruited to p16(INK4a) promoter, and the histone acetyltransferase domain of p300 participated in p16(INK4a) activation through inducing hyperacetylation of histone H4 at p16(INK4a) gene. These data suggest that p300 plays a critical role in transcriptional regulation of p16(INK4a) and in cell cycle arrest.