An unknown and almost fully encrypted letter written in 1547 by Emperor Charles V to his ambassador at the French Court, Jean de Saint-Mauris, was identified in a public library, the Bibliothèque Stanislas (Nancy, France). As no decryption of this letter was previously published or even known, a team of cryptographers and historians gathered together to study the letter and its encryption system. First, multiple approaches and methods were tested in order to decipher the letter without any other specimen. Then, the letter has now been inserted within the whole correspondence between Charles and Saint-Mauris, and the key has been consolidated thanks to previous key reconstructions. Finally, the decryption effort enabled us to uncover the content of the letter and investigate more deeply both cryptanalysis challenges and encryption methods.