Senescence is triggered by various cellular stresses that result in genomic lesions and DNA damage response activation. However, the role of chromatin and DNA replication in senescence induction remains elusive. Here we show that downregulation of p300 histone acetyltransferase activity induces senescence by a mechanism that is independent of the activation of p53, p21(CIP1) and p16(INK4A). This inhibition leads to a global H3, H4 hypoacetylation, initiating senescence-associated heterochromatic foci formation during S phase, together with a global decrease in replication fork velocity, and alteration of DNA replication timing. This replicative stress occurs without DNA damage and checkpoint activation, but results in a robust G2/M cell cycle arrest, within only one cell cycle. These results provide new insights into the control of S-phase progression by p300, and identify an unexpected chromatin-dependent alternative mechanism for senescence induction, which could possibly be exploited to treat cancer by senescence induction without generating further DNA damage.