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 NH3–N, branched fatty acids, butyrate and acetic:propionic ratio, and decreased plasma δ15N. The supplement promoted bacterial populations involved in hemicellulose and pectin degradation and ammonia assimilation: Bacteroidales BS11, Cyanobacteria, and Prevotella spp. During the dry season, fibrolytic populations were promoted: the bacteria Fibrobacter, Cyanobacteria and Kiritimatiellaeota groups; the fungi Cyllamyces; and the protozoa Ostracodinium. The wet season increased the abundances of rumen protozoa and fungi populations, with increases of bacterial families Lachnospiraceae, Ruminococcaceae, and Muribaculaceae; the protozoa Entodinium and Eudiplodinium; the fungi Pecoramyces; and the archaea Methanosphera. 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.