Abstract Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Hdm2-p53 interface or HdmX-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Hdm2–HdmX which is mediated by RING–RING interaction has recently appeared as a promising novel anticancer approach. Targeting the E3 ligase of Hdm2-HdmX should overcome HdmX-driven resistance, which is found in many cancers overexpressing HdmX, to p53-Hdm2 antagonists such a nutlins. Cyclotides are a novel class of micro-proteins (≈30-40 residues long) with a unique topology containing a head-to-tail cyclized backbone structure further stabilized by three disulfide bonds that form a cystine knot. This unique molecular framework makes them exceptionally stable to physical, chemical, and biological degradation compared to linear peptides of similar size. The cyclotides are also highly tolerant to sequence variability, aside from the conserved residues forming the cystine knot, orally bioavailable and able to cross cellular membranes to modulate intracellular protein-protein interactions (PPIs) both in vitro and in vivo. These unique properties make them ideal scaffolds for many biotechnological applications, including drug discovery. We report the discovery of a novel cyclotide, MCo-52-2, able to antagonize efficiently the interaction between the RING domains of Hdm2 and HdmX and show p53-specific anticancer activity in several cancer cell lines both in vitro and in vivo. We will also present different strategies to improve the cellular uptake and pharmacokinetic profiles of bioactive cyclotides. Citation Format: Julio A Camarero. Therapeutic targeting of Hdm2/HdmX E3 ligase with bioactive cyclotides [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A134. doi:10.1158/1535-7163.TARG-19-A134