An in vitro study was conducted to investigate the effect of different concentrations of Euglena (Euglena gracilis) on CH4 production, dry matter (DM) digestibility, volatile fatty acid (VFA) and ammonia N(NH3-N) concentration as well as on the protozoa population. The treatments considered were Euglena at concentrations of 0.0, 50, 100, 200, 400 and 1000g/kg dry matter (DM) of the substrate (60:40 forage: concentrate ratio) incubated for 24 and 96h using an in vitro continuous gas production and in vitro two-stage digestion procedure, respectively. The data were subjected to polynomial regression analysis. Methane emissions (ml/g DM) decreased at an increasing rate, generally with increasing concentration of Euglena but also exhibited quadratic (P<0.001) and cubic (P<0.001) effects while NH3-N (mg/ml) concentration increased at an increasing rate (linear P<0.001; quadratic P=0.001; cubic P=0.024). Total VFA concentration (mmol/l) decreased significantly (P<0.001), when the substrate was totally replaced by Euglena. There was a linear (P<0.001) and cubic (P=0.047) reduction in protozoa population as the concentration of Euglena increased. In vitro DM digestibility was improved (linear P=0.003; quadratic P=0.04; cubic P<0.001). These findings demonstrate that Euglena at concentration of 100g/kg DM reduce CH4 emissions by 9.1% and improve DM digestibility by 14.3%. However, when the concentration of Euglena increases from 100g/kg DM, while further reducing CH4 emissions, have negative effect on NH3-N concentration, protozoa population and VFA concentration.