Abstract The objective of this study was to investigate the effects of a recombinant fibrolytic enzyme (RFE; xylanase XYL10C) on ruminal pH and fermentation characteristics, total tract digestibility and nitrogen balance in beef heifers. The experiment was a cross-over design with two treatments and two periods, using eight ruminal cannulated beef heifers. The treatments were a basal diet containing 60% barley silage, 30% barley straw and 10% supplement (DM basis) without (control) or with RFE. The RFE XYL10C was selected specifically for ruminants using a high throughput in vitro micro assay and was sprayed onto the barley straw at a rate of 6.6 × 104 IU/kg DM 24 h before feeding. The diet was prepared daily using a data ranger. Each period was 21 d, with a 14-d adaptation and 7-d sample collection period. The digestibility and nitrogen balance were measured by total fecal and urine collection. Data were analyzed using the PROC MIXED procedure of SAS with fixed effects of treatment, and random effects of period and heifer. Feed intake (8.9 kg/d) and total tract digestibility of DM (65.5%), NDF (57.2%) and ADF (51.2%) were not affected by RFE. Ruminal pH including mean (6.52), minimum (5.79), maximum (7.05), and duration of pH < 5.8 (0.64, h/d) did not differ between treatments. No treatment effects were observed for total VFA concentration (106 mM), molar proportion of acetate (68.2%) and propionate (17.5%) and acetate to propionate ratio (3.94). However, adding RFE vs. control tended (P < 0.08) to increase ruminal NH3-N concentration (5.75 vs. 5.23 mM) and endoglucanase activity (44.3 vs. 40.7 µmol glucose/min/mL wet rumen contents). There were no differences in nitrogen utilization and rumen microbial protein synthesis (90.7 g/d) between treatments. The results indicate that RFE XYL10C did not improve fiber digestion in heifers fed a high forage diet.