Abstract Compelling evidence demonstrated that melatonin increases p53 activity in cancer cells. p53 undergoes acetylation to be stabilized and activated for driving cells destined for apoptosis/growth inhibition. Over‐expression of p300 induces p53 acetylation, leading to cell growth arrest by increasing p21 expression. In turn, p53 activation is mainly regulated in the nucleus by MDM 2. MDM 2 also acts as E3 ubiquitin ligase, promoting the proteasome‐dependent p53 degradation. MDM 2 entry into the nucleus is finely tuned by two different modulations: the ribosomal protein L11, acts by sequestering MDM 2 in the cytosol, whereas the PI 3K‐AkT‐dependent MDM 2 phosphorylation is mandatory for MDM 2 translocation across the nuclear membrane. In addition, MDM 2‐dependent targeting of p53 is regulated in a nonlinear fashion by MDM 2/ MDMX interplay. Melatonin induces both cell growth inhibition and apoptosis in MCF 7 breast cancer cells. We previously reported that this effect is associated with reduced MDM 2 levels and increased p53 activity. Herein, we demonstrated that melatonin drastically down‐regulates MDM 2 gene expression and inhibits MDM 2 shuttling into the nucleus, given that melatonin increases L11 and inhibits Akt‐ PI 3K‐dependent MDM 2 phosphorylation. Melatonin induces a 3‐fold increase in both MDMX and p300 levels, decreasing simultaneously Sirt1, a specific inhibitor of p300 activity. Consequently, melatonin‐treated cells display significantly higher values of both p53 and acetylated p53. Thus, a 15‐fold increase in p21 levels was observed in melatonin‐treated cancer cells. Our results provide evidence that melatonin enhances p53 acetylation by modulating the MDM 2/ MDMX /p300 pathway, disclosing new insights for understanding its anticancer effect.