Significance Brucella species are capable of infecting a range of mammals, including humans, with relative host specificity. We hypothesized that metabolism is instrumental in host adaptation and compared the central metabolism of seven species. We demonstrate the existence of two distinct groups, including one overlapping with the classical zoonotic brucellae of domestic livestock that exclusively use the pentose phosphate pathway. Species from the second group rely mostly on the Entner–Doudoroff pathway instead. This metabolic dichotomy arose after the acquisition of two newly identified EDP-inactivating mutations. This selected trait seems to be linked to bacterial pathogenicity in mice. The data fit the hypothesis that Brucella has undergone a stepwise metabolic evolution in domestic hosts that might also apply to other pathogens.