The C-terminal portion of the E protein, known as stem, is conserved among flaviviruses and is an important target to peptide-based antiviral strategies. Since the dengue (DENV) and Zika (ZIKV) viruses share sequences in the stem region, in this study we evaluated the cross-inhibition of ZIKV by the stem-based DV2 peptide (419–447), which was previously described to inhibit all DENV serotypes. Thus, the anti-ZIKV effects induced by treatments with the DV2 peptide were tested in both in vitro and in vivo conditions. Molecular modeling approaches have demonstrated that the DV2 peptide interacts with amino acid residues exposed on the surface of pre- and postfusion forms of the ZIKA envelope (E) protein. The peptide did not have any significant cytotoxic effects on eukaryotic cells but efficiently inhibited ZIKV infectivity in cultivated Vero cells. In addition, the DV2 peptide reduced morbidity and mortality in mice subjected to lethal challenges with a ZIKV strain isolated in Brazil. Taken together, the present results support the therapeutic potential of the DV2 peptide against ZIKV infections and open perspectives for the development and clinical testing of anti-flavivirus treatments based on synthetic stem-based peptides.