Objective: This research was arranged to explore the effect of supplementation of a combination of Lactobacillus plantarum and Saccharomyces cerevisiae as a new probiotic in fermented rice straw-based rations on in vitro digestibility and ruminal characteristics. Materials and Methods: A randomized group design with 3 types of treatment and 4 replications as a group was used in this study. A probiotic inoculum containing L. plantarum and S. cerevisiae with 1 × 1010 colony-forming unit (CFU)/ml. Treatments were followed by: P1 = complete rations without probiotics (control), P2 = P1 supplemented 0.5% probiotics, and P3 = P1 supplemented 1% probiotics. Substrate complete rations were based on the fermented rice straw and concen¬trate (60%:40%). Parameters of digestibility and rumen fermentation products were determined after 48 h of incubation. Results: Probiotics supplemented with fermented rice straw-based rations significantly increased (p < 0.05) digestibility and rumen characteristics in vitro. Supplementation with 1% probiotics (P3) produces the highest digestibility compared to other treatments: in-vitro dry matter digestibil¬ity (IVDMD) (55%), in-vitro organic matter digestibility (IVOMD) (58.28%), in-vitro crude protein digestibility (IVCPD) (84.42%), in-vitro acid detergent fiber digestibility (IVADFD) (53.99%), in-vitro neutral detergent fiber digestibility (IVNDFD) (58.39%), and in-vitro cellulose digestibility (IVCLD) (67.12%). Rumen pH (6.76–6.80) did not change significantly (p > 0.05) due to supplemented probiotics. Probiotic supplementation in rations significantly (p < 0.05) increased the content of NH3 and total volatile fatty acid (VFA). Supplementation with 1% probiotic (P3) showed the highest concentration of NH3 (26.56 mg/100 ml) and was also followed by the total VFA (115.75 mM) compared to the control (22.59 mg/100 ml and 103.00 mM, respectively). Conclusion: Supplementation of 1% probiotics (combination of L. plantarum and S. cerevisiae) containing 1 × 1010 CFU/ml in fermented rice straw-based rations increases nutrient digestibility, that is, IVDMD, IVOMD, IVCPD, IVADFD, IVNDFD, and IVCLD, and also increases rumen fermenta¬tion, which is the concentration of NH3 and total VFA.