The use of cereals as forage crops is limited due to the high lignin content in the cell walls reducing nutrient digestibility. Recent research has focused on reducing lignification in forage crops through gene mutations. This study investigated the ruminal fermentation characteristics of a barley mutation (orange lemma), which is associated with a lower lignin content, using the in vitro ruminal fermentation system (RUSITEC). Two-rowed spring barley cv. Optic' and its ethyl methane sulfonate (EMS)-induced orange lemma (rob1) mutant line were harvested at both stem elongation and early fruit development and incubated in the RUSITEC system. Gas production, concentrations of short-chain fatty acids (SCFA) and ammonia and the nutrient degradation of the plants after 48 h incubation were investigated. Additional samples were analysed for microbial composition using MiSeq sequencing technology. In general, acid detergent lignin (ADL) was higher at early grain filling than stem elongation. ADL was lower in the mutant line than in the wild type at both stem elongation (13.9% vs 18.5%) and early grain development (26.0% vs 28.6%; dry matter basis). This was reflected in increased ruminal degradation of neutral detergent fibre (61.7% vs 53.7%; P < 0.001) when harvested at stem elongation, but not at the later stage. In contrast, methane formation was lower with rob1 than Optic' (P = 0.002), especially when harvested at stem elongation. No difference was seen in protein degradation between the barley genotypes. The fermentation SCFA profile did not differ between barley genotypes when harvested at stem elongation, but at early fruit development more acetate and less butyrate was produced with rob1. Microbial species richness was lower when barley was incubated at stem elongation compared to fruit development (P < 0.001), which was especially pronounced with rob1 (P = 0.026). The abundance of Bacteroidetes, Synergistetes and Tenericutes was lower when plants harvested at early grain development were incubated compared to the stem elongation stage, whereas the abundance of Cyanobacteria, Elusimicrobia, Fusobacteria, Lentisphaerae, Proteobacteria, Verrucomicrobia and WPS-2 was higher (P < 0.001). In conclusion, most fermentation parameters were affected by vegetation stage and related changes in nutrient composition. However, additional effects of barley genotype were seen on the rumen microbial community structure, SCFA profile and methane production.