| Summary: | This study aimed to characterize the rumen microbiota structure of cattle grazing in tropical rangelands throughout seasons and their responses in rumen ecology and productivity to a N-based supplement during the dry season. Twenty pregnant heifers grazing during the dry season of northern Australia were allocated to either N-supplemented or un-supplemented diets and monitored through the seasons. Rumen fluid, blood, and feces were analyzed before supplementation (mid-dry season), after two months supplementation (late-dry season), and post supplementation (wet season). Supplementation increased average daily weight gain (ADWG), rumen NH<sub>3</sub>–N, branched fatty acids, butyrate and acetic:propionic ratio, and decreased plasma δ<sup>15</sup>N. The supplement promoted bacterial populations involved in hemicellulose and pectin degradation and ammonia assimilation: <i>Bacteroidales BS11</i>, Cyanobacteria, and <i>Prevotella</i> spp. During the dry season, fibrolytic populations were promoted: the bacteria <i>Fibrobacter</i>, Cyanobacteria and Kiritimatiellaeota groups; the fungi <i>Cyllamyces</i>; and the protozoa <i>Ostracodinium</i>. The wet season increased the abundances of rumen protozoa and fungi populations, with increases of bacterial families <i>Lachnospiraceae</i>, <i>Ruminococcaceae</i>, and <i>Muribaculaceae</i>; the protozoa <i>Entodinium</i> and <i>Eudiplodinium</i>; the fungi <i>Pecoramyces</i>; and the archaea <i>Methanosphera</i>. In conclusion, the rumen microbiota of cattle grazing in a tropical grassland is distinctive from published studies that mainly describe ruminants consuming better quality diets.
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