Piscirickettsia salmonis is a facultative Gram-negative intracellular bacterium that produces piscirickettsiosis, disease that causes a high negative impact in salmonid cultures. The so-far-unidentified nutritional requirements have hindered its axenic culture at laboratory and industrial scales for the formulation of vaccines. The present study describes the development of a defined culture medium for P. salmonis. The culture medium was formulated through rational design involving auxotrophy test and statistical designs of experiments, considering the genome-scale metabolic reconstruction of P. salmonis reported by our group. The whole optimization process allowed for a twofold increase in biomass and a reduction of about 50% of the amino acids added to the culture medium. The final culture medium contains twelve amino acids, where glutamic acid, threonine and arginine were the main carbon and energy sources, supporting 1.65 g/L of biomass using 6.5 g/L of amino acids in the formulation. These results will contribute significantly to the development of new operational strategies to culture this bacterium for the production of vaccines.