The combined addition of branched-chain volatile fatty acids (BCVFAs) and folic acid (FA) could improve growth performance and nutrient utilization by stimulating ruminal microbial growth and enzyme activity. This study was conducted to evaluate the effects of BCVFA and FA addition on growth performance, ruminal fermentation, nutrient digestibility, microbial enzyme activity, microflora and excretion of urinary purine derivatives (PDs) in calves. Thirty-six Chinese Holstein weaned calves (60 ± 5.4 days of age and 107 ± 4.7 kg of BW) were assigned to one of four groups in a randomized block design. Treatments were control (without additives), FA (with 10 mg FA/kg dietary DM), BCVFA (with 5 g BCVFA/kg dietary DM) and the combined addition of FA and BCVFA (10 mg/kg DM of FA and 5 g/kg DM of BCVFA). Supplements were hand-mixed into the top one-third of total mixed ration. Dietary concentrate to maize silage ratio was 50 : 50 on a DM basis. Dietary BCVFA or FA addition did not affect dry matter intake but increased average daily gain (ADG) and feed conversion efficiency. Ruminal pH and ammonia N were lower, and total volatile fatty acids (VFAs) concentration was higher for BCVFA or FA addition than for control. Dietary BCVFA or FA addition did not affect acetate proportion but decreased propionate proportion and increased acetate to propionate ratio. Total tract digestibility of DM, organic matter, CP and NDF was higher for BCVFA or FA addition than for control. Dietary BCVFA or FA addition increased activity of carboxymethyl cellulase and cellobiase, population of total bacteria, fungi, Ruminococcus albus, R. flavefaciens, Fibrobacter succinogenes and Prevotella ruminicola as well as total PD excretion. Ruminal xylanase, pectinase and protease activity and Butyrivibrio fibrisolvens population were increased by BCVFA addition, whereas population of protozoa and methanogens was increased by FA addition. The BCVFA × FA interaction was significant for acetate to propionate ratio, cellobiase activity and total PD excretion, and these variables increased more with FA addition in diet without BCVFA than in diet with BCVFA. The data indicated that supplementation with BCVFA or FA increased ADG, nutrient digestibility, ruminal total VFA concentration and microbial protein synthesis by stimulating ruminal microbial growth and enzyme activity in calves.