Brucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915 T , showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.