A completely randomized experiment for planting highland barley in 36 field plots of the Lhasa Agricultural Experiment Station was applied to investigate the effect of urea nitrogen (N) fertilization levels of 0 (control), 156, 258, 363, 465 and 570 kg/ha on nutrient accumulation, in vitro rumen gas production and fermentation characteristics of highland barley straw (HBS). Each urea application was divided into three portions of 0.4, 0.3 and 0.3 and sequentially fertilized at seeding (growth stage (GS) 0), stem elongation (GS 32) and heading (GS 49), respectively. The maturity stage lasted 5–13 days longer in response to the urea N fertilization compared with the control. After removing grains, HBS biomass was harvested at maturity. The biomass yields of leaf, stem, straw and grain were increased quadratically with increasing urea N fertilization, and HBS and grain yields peaked at the estimated urea N fertilization levels of 385 and 428 kg/ha, respectively. The increase of urea N fertilization increased the accumulation of crude protein, cellulose and lignin, and decreased the content of ash and hemicellulose in HBS, resulting in a decrease of the energy content available to be metabolized. After incubating HBS for 72 h with rumen fluids from lactating cows, the urea N fertilization decreased in vitro dry matter disappearance and cumulative gas production, and slightly altered fermentation end-gas composition. Urea N fertilization decreased microbial volatile fatty acid production, but did not alter the ratio of lipogenic acetate and butyrate to glucogenic propionate. In a brief, the current urea N fertilization strategy promoted the growth of the highland barley and increased biomass yield, protein and cellulose accumulation of HBS. A urea N fertilization level ⩽385 kg/ha could be sufficient for growth of highland barley in Tibet without a consequent nutritive reduction in ruminal digestion.