| Summary: | The objective of this research is to investigate the effect of yeast (<i>Saccharomyces cerevisiae</i>) adding and roughage-to-concentrate ratio (R:C ratio) on nutrients utilization, rumen fermentation efficiency, microbial protein synthesis, and protozoal population in Thai native beef cattle. Four Thai native beef cattle, weighing an average of 120 ± 10 kg live weight, were randomly assigned to four dietary treatments using a 2 × 2 factorial arrangement in a 4 × 4 Latin square design. Factor A was the level of roughage-to-concentrate ratio (R:C ratio) at 60:40 and 40:60; factor B was the levels of live yeast (LY) supplementation at 0 and 4 g/hd/d; urea–calcium-hydroxide-treated rice straw were used as a roughage source. Findings revealed that total intake and digestibility of dry matter (DM), organic matter (OM), and crude protein (CP) were increased (<i>p</i> < 0.05) by both factors, being greater for steers fed a R:C ratio of 40:60 supplemented with 4 g LY/hd/d. Ruminal ammonia nitrogen, total volatile fatty acid (VFA), and propionate (C<sub>3</sub>) were increased (<i>p</i> < 0.05) at the R:C ratio of 40:60 with LY supplementation at 4 g/hd/d, whereas rumen acetate (C<sub>2</sub>) and the C<sub>2</sub> to C<sub>3</sub> ratio were decreased (<i>p</i> < 0.05). With a high level of concentrate, LY addition increased total bacterial direct counts and fungal zoospores (<i>p</i> < 0.05), but decreased protozoal populations (<i>p</i> < 0.05). High-concentrate diet and LY supplementation increased nitrogen absorption and the efficiency of microbial nitrogen protein production. In conclusion, feeding beef cattle with 4 g/hd/d LY at a R:C ratio of 40:60 increased C<sub>3</sub> and nutritional digestibility while lowering protozoal population.
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