Since the development of the yellow fever (YF) vaccine 17D, in the 1930’s, around 400 million people have been vaccinated with a level of protection of more than 98%. The 17D has been used as a viral vector for the insertion of heterologous epitopes, seeking after new vaccines. To allow the expression of these antigens at the external surface of the virion particle, we here describe a new site of insertion into the E protein of the YF virus. Based on the know crystal structures, the new site (E₀F₀ loop) exhibit a higher than average B-factors and is located on the external surface of the E protein, within domain I. In the trimeric structures of flavivirus E protein this pattern is also repeated, suggesting inherent high flexibility of this region. The E₀F₀ loop is located at the proximity of the holes, between subunits of the dimer, surrounded by a large volume of solvent which provides ample space for accommodating the insert. Three cloning strategies involving distinct mutations, deletions or duplication of E₀F₀ loop residues were applied for an insertion of a foreign repetitive epitope - (NANP)₃. This antigenic determinant belongs to the central domain of Plasmodium falciparum CS protein. Growth curves in Vero cells showed different profiles of replication capability of the three distinct insertion-bearing viruses compared to the control YF virus. In addition, the recombinant viruses were less virulent than the vaccinal strain in murine model. The mortality rates varied from 5 to 40%, with an average survival time of 13 days. These results were statistically significant (P<0.001) compared to the values obtained with the vaccine inoculation (100% mortality and AST of 11 days). The recombinant viruses showed immune response against the YF virus and the foreign epitope. Besides this, 90% of these animals survived to an intra-cerebral challenge with YF 17D virus. These findings highlight the potential use of this site for the development of new live attenuated 17D virus-based vaccines.