The effect of Nigella sativa L. as saponin sources on in vitro rumen fermentation, enzyme activity, and nutrients digestibility was investigated in this study. The diet consisted of 70% Napier Grass (Pennisetum purpureum) and 30% wheat pollard containing 0%, 0.2%, 0.4%, and 0.6% saponin, respectively. In vitro fermentation was conducted using Menke and Steingass gas production and two-stage Tilley and Terry. The acquired data were subjected to variance analysis and Duncan's Multiple Range Test (DMRT). The addition of Nigella sativa L. meal significantly decreases (P<0.05) methane (digested organic matter), protozoa population, and increased microbial protein but does not affect (P>0.05) pH, ammonia concentration, and volatile fatty acids (VFA) production. CMCase enzyme activity reduced (P<0.05) as saponin levels increased, but amylase and protein of enzyme were unaffected (P>0.05). Saponin reduced protein digestibility in the rumen, but there was no difference in dry matter (DM) or organic matter (OM) when compared to the control (P>0.05). Post-rumen nutrients digestibility did not affect by the saponin diet (P>0.05). In conclusion, saponins reduced protozoa population, CH 4 production, increased microbial protein, and improve rumen protein digestibility. Therefore, we recommend the use of 0.4% saponins Nigella sativa L. to reduce CH 4 production without affecting rumen fermentation.