<jats:p>The genus <jats:italic> <jats:named-content content-type="genus"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.8042" xlink:type="simple">Prevotella</jats:ext-link> </jats:named-content> </jats:italic> plays an important role in polysaccharide degradation and fermentation in the rumen. To further understand the function of the phylogenetically diverse genus <jats:italic> <jats:named-content content-type="genus"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.8042" xlink:type="simple">Prevotella</jats:ext-link> </jats:named-content> </jats:italic>, it is necessary to explore the individual characteristics at the species level. In this study, Gram-negative anaerobic bacterial strains isolated from the rumen of Holstein cows were identified. Strain R5019<jats:sup>T</jats:sup> was classified within the genus <jats:italic> <jats:named-content content-type="genus"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.8042" xlink:type="simple">Prevotella</jats:ext-link> </jats:named-content> </jats:italic> based on 16S rRNA gene sequence-based phylogenetic analysis. The values of 16S rRNA gene sequence similarity, average nucleotide identity and digital DNA–DNA hybridization between strain R5019<jats:sup>T</jats:sup> and its phylogenetically nearest species <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.9639" xlink:type="simple">Prevotella multisaccharivorax</jats:ext-link> </jats:named-content> </jats:italic> PPPA20<jats:sup>T</jats:sup> were 89.8, 82.6, and 29.3 %, respectively. The genome size of R5019<jats:sup>T</jats:sup> was estimated to be ca. 4.19 Mb with a genomic G+C content of 49.5 mol%. The major cellular fatty acids and menaquinones were C<jats:sub>15 : 0</jats:sub> anteiso and C<jats:sub>17 : 0</jats:sub> anteiso and MK-11 and MK-12, respectively. Succinate, lactate, malate, acetate and formate were produced as the fermentation end products using glucose. Based on phylogenetic, physiological, biochemical and genomic differences between 11 strains and other phylogenetically related <jats:italic> <jats:named-content content-type="genus"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.8042" xlink:type="simple">Prevotella</jats:ext-link> </jats:named-content> </jats:italic> species, a novel species, <jats:italic>Prevotella lacticifex</jats:italic> sp. nov., is proposed within the genus <jats:italic> <jats:named-content content-type="genus"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.8042" xlink:type="simple">Prevotella</jats:ext-link> </jats:named-content> </jats:italic>. The type strain is R5019<jats:sup>T</jats:sup> (=JCM 34664<jats:sup>T</jats:sup>=DSM 112675<jats:sup>T</jats:sup>).</jats:p>