Accumulation of senescent cells during aging contributes to chronic inflammation and age-related diseases. While senescence is associated with profound alterations of the epigenome, a systematic view of epigenetic factors in regulating senescence is lacking. Here, we curated a library of short hairpin RNAs for targeted silencing of all known epigenetic proteins and performed a high-throughput screen to identify key candidates whose downregulation can delay replicative senescence of primary human cells. This screen identified multiple new players including the histone acetyltransferase p300 that was found to be a primary driver of the senescent phenotype. p300, but not the paralogous CBP, induces a dynamic hyper-acetylated chromatin state and promotes the formation of active enhancer elements in the non-coding genome, leading to a senescence-specific gene expression program. Our work illustrates a causal role of histone acetyltransferases and acetylation in senescence and suggests p300 as a potential therapeutic target for senescence and age-related diseases. Display omitted •High-throughput screening identifies key epigenetic proteins driving senescence•Depletion of the histone acetyltransferase p300 delays senescence•p300 induces the formation of de novo super enhancers in senescence•Depletion of p300 suppresses senescence-related gene expression Epigenetic dysregulation is a hallmark of senescence and aging. In this article, Sen et al. have identified the histone acetyltransferase p300 as a key protein regulating senescence from a high-throughput screen. p300 induces formation of new super enhancers that drive senescence-related gene expression. p300 is an attractive candidate for anti-aging therapy.