EPI-X4 is a natural peptide antagonist of CXCR4, an established drug target in inflammatory diseases and cancer. Advanced derivatives of EPI-X4, such as EPI-X4 JM#21, have shown promising therapeutic effects in animal models of CXCR4-associated diseases but suffer from poor stability in blood. We here aimed to design and characterize EPI-X4 analogs that effectively antagonize CXCR4 while remaining stable in human plasma. We found that EPI-X4 analogs are stable against degradation by endopeptidases. However derivatives are are prone to degradation by exopeptidases which target the peptides’ N- but not the C-terminus. Modifications of the peptide N-terminus by introducing D-amino acids or acetyl residues resulted in EPI-X4 derivatives with greatly enhanced plasma stability. The identified lead candidates EPI-X4 JM#29, JM#173, and JM#174, which contain D-amino acids L or I at position 1, were as active in binding and antagonizing CXCR4 as EPI-X4 JM#21, and remained active even after 8 hours of incubation in plasma. Molecular dynamics simulations showed that the binding mode of these stabilized EPI-X4 derivatives to CXCR4 is similar to the binding of JM#21. The peptides establish conserved interactions with acidic residues in the minor subpocket and the extracellular loops 1 and 2 of the receptor. None of the novel EPI-X4 leads showed any signs of toxicity in zebrafish embryos, paving the way for further evaluation of the pharmacokinetic and therapeutic properties of this new generation of EPI-X4 analogs in rodent models.