The current experiment was conducted to evaluate the effects of different dietary protein levels and rumen-protected folic acid (RPFA) supplementation on ruminal fermentation, microbial enzyme activity, bacterial populations and urinary excretion of purine derivatives (PD) in growing beef steers. Low-protein (LP) or high-protein (HP) diets were fed to eight ruminally cannulated first-generation cross-bred (Blonde d'Aquitaine × Simmental) beef steers with or without RPFA supplementation. Steers were fed a total mixed ration, and dietary concentrate to maize silage ratio was 50 : 50 (dry matter (DM) basis). No interaction between dietary crude protein (CP) levels and RPFA supplementation was observed during the experiment. Ruminal pH was unaffected by RPFA supplementation, but decreased with increasing dietary CP levels. Ruminal total volatile fatty acid concentration increased with increasing dietary CP levels or RPFA supplementation. Molar proportion of acetate increased with RPFA supplementation, but tended to decrease with increasing dietary CP levels. The proportion of propionate decreased with RPFA supplementation, but was unaffected by dietary CP levels. As a result, the acetate to propionate ratio increased with RPFA supplementation, but tended to be lower for the HP diets than the LP diets. Ammonia-nitrogen content decreased with RPFA supplementation, but increased with increasing dietary CP levels. In situ ruminal degradability of maize straw and concentrate increased with increasing dietary CP levels or RPFA supplementation. Microbial enzyme (carboxymethyl-cellulase, cellobiase, xylanase, pectinase, α-amylase and protease) activity, bacterial populations (Ruminococcus albus, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, Prevotella ruminicola, Fibrobacter succinogenes and Ruminobacter amylophilus) and urinary PD excretion increased with increasing dietary CP levels or RPFA supplementation. The current study showed that increasing dietary CP levels from 109·1 to 130·7 g/kg DM or supplementing 75 mg RPFA improved ruminal fermentation and microbial protein synthesis by increasing bacterial population and microbial enzyme activity.