Brucella ovisis a non-zoonotic roughBrucellathat causes genital lesions, abortions and increased perinatal mortality in sheep and is responsible for important economic losses worldwide. Research on virulence factors ofB. ovisis necessary for deciphering the mechanisms that enable this facultative intracellular pathogen to establish persistent infections and for developing a species-specific vaccine, a need in areas where the cross-protecting ovine smoothB. melitensisRev1 vaccine is banned. Although severalB. ovisvirulence factors have been identified, there is little information on its metabolic abilities and their role in virulence. Here, we report that deletion of pyruvate phosphate dikinase (PpdK, catalyzing the bidirectional conversion pyruvate -cc; phosphoenolpyruvate) inB. ovisPA (virulent and CO2-dependent) impaired growth in vitro. In cell infection experiments, although showing an initial survival higher than that of the parental strain, thisppdKmutant was unable to multiply. Moreover, when inoculated at high doses in mice, it displayed an initial spleen colonization higher than that of the parental strain followed by a marked comparative decrease, an unusual pattern of attenuation in mice. A homologous mutant was also obtained in aB. ovisPA CO2-independent construct previously proposed for developingB. ovisvaccines to solve the problem that CO2-dependence represents for large scale production. This CO2-independentppdKmutant reproduced the growth defect in vitro and the multiplication/clearance pattern in mouse spleens, and is thus an interesting vaccine candidate for the immunoprophylaxis ofB. ovisovine brucellosis.