Targeting the heterodimer MDM2/MDM4 is a novel and effective route for the reactivation of wild-type p53 in human tumors with reduced toxicity in nontransformed cells. To improve the therapeutic potential of peptides that interfere with MDM4 binding to MDM2, we demonstrated the tumor-suppressive activity of a short peptide (Pep3S), which is composed of the last five amino acids of the MDM4 protein. Compared to longer peptides (previously identified), Pep3S binds MDM2 with high affinity, increases p53-dependent cell death in 2D and 3D colorectal cancer models, and is more efficacious in suppressing xenograft tumor growth. Furthermore, its encapsulation in poly (lactic-co-glycolic acid) (PLGA) nanoparticles potentiated and prolonged its activity. A p53-specific target gene array revealed an uncommon p53 signature, with Pep3S leading to p53-mediated repression of a subset of p53 targets. Comparative analysis indicated that this repression is driven by p53-mediated activation of miR-34a, which is functional in Pep3S-induced cell death. Of note, unlike other p53-reactivating molecules, Pep3S led to significant downregulation of the cell cycle inhibitor CDKN1A/p21, one of the best-characterized p53-targets. Genetic manipulation of MDM4 demonstrated the requirement of the dissociated protein for p21 downregulation, whereas the miR-34a signature was not altered. At odds with Nutlin-3a, the proliferation status of nontumor muscle and lymphoblastoid cells was not altered by Pep3S. These data indicate that targeting the MDM2/MDM4 interaction region provides a different route for wild-type p53 reactivation in human tumors, potentially reducing toxicity to proliferating nontumor tissue. The development of a PLGA/Pep3S formulation represents a promising approach for therapeutic purposes.