Abstract: Background and Objective: Tannins have the ability to bind protein and can act to increase animal productivity by rendering protein inactive in the rumen and releasing it post-rumen for use by the animal for meat or milk production.This study was to determine the effect of supplementation of tannin from two different sources at different doses to ammoniated oil palm frond on in vitro rumen fermentation characteristics. Source of tannin is Gambier Leaves Waste (GLW) from Payakumbuh and Painan, two different districts in West Sumatera province. Doses of GLW on dry matter bases. Materials and Methods: The experiment was arranged in a block randomized design with seven treatments and three replications. The treatments were A = oil palm frond was treated with 4% urea, B1 = A+10% GLW Payakumbuh, B2 = A+ 15% GLW Payakumbuh, B3 = A+20% GLW Payakumbuh, C1 = A+10% GLW Painan, C2 = A+ 15% GLW Painan and C3 = A+20% GLW Painan. Variables measured were methane production, DM, OM, NDF and ADF digestibility, concentration of NH3-N, partial VFAs, microbial protein synthesis, population of rumen bacteria and protozoa and ruminal fluid pH. Data was analyzed using analysis of variance (ANOVA) and differences among means were tested using Duncan. Results: The results showed that the supplementation of GLW were significantly (p<0.05) increased degradability, fermentability and reduce methane gas production. The treatment had no significant effect (p>0.05) on ruminal fluid pH, propionate production and population of bacteria. The DM digestibility increased from 48.45 (Treatment A)-52.95% (Treatment B2) and OM digestibility 51.34 (Treatment A)-57.30% (Treatment B2). The concentration of VFAs increased from 71.00-95.78 mM. Molar proportion of VFAs was shifted from acetate to propionate production and reduced the ratio of acetate to propionate. The rumen pH with supplementation of tannins is relatively more stable. Methane production decreased from 27.22 (Treatment A)-12.67 mM (Treatment B2) and to 15.13 mM (Treatment C1). The methane production reduced to 53% (Treatment B2) and 45% (Treatment C1) compare control. Conclusion: These results showed that 15% GLW Payakumbuh and 10% GLW Painan was suitable to be used as doses and source of tannins but the supplementation of 15% GLW Payakumbuh give the best results on digestibility and in reducing methane gas production.