The tumor suppressor p53 plays a key role in regulation of the cell cycle, apoptosis and senescence in response to various stresses. We screened a library of 7920 chemical compounds for the p53 activator and identified N-[2-(dimethylamino)ethyl]-2,3-dimethyl-4-oxo-4H-pyrido[1,2-a]thieno[2,3-d]pyrimidine-9-carboxamide (PTP), which significantly increased p53-mediated reporter activity in colorectal cancer cells. PTP was found to induce p53 protein and activated transcription of downstream genes, such as p21 and PUMA, in HCT116 cells, leading to growth delay, G1-phase cell cycle arrest, cell senescence and cell death. Proximity ligation assay revealed that PTP weakened the interaction between p53 and murine double minute 2 (MDM2) in situ, thereby inhibiting MDM2-mediated p53 degradation. Although DNA damage has been known to promote phosphorylation of p53 and MDM2, thereby preventing their interaction and stabilizing p53, PTP did not cause DNA damage or activate any DNA damage response signaling. Instead, phosphorylation of p53 was mediated by Erk1/2 MAP kinase. In addition, PTP induced acetylation of p53 at Lys382 in a p300-dependent manner, but sirtuin (SIRT)1 and histone deacetylase (HDAC)1, a well-known p53-regulating deacetylase, were not involved. In the present study, the novel anticancer agent PTP was shown to cause the accumulation of p53 by inducing multiple post-translational modifications, as well as cell cycle arrest, senescence and cell death.