Abstract: Background and Objective: Probiotics are widely used in ruminant production, but information about the potential of Lactobacillus plantarum (L. plantarum) as a probiotic for ruminants is still limited. The aim of this research was to select L. plantarum strains as a probiotic for ruminants and to determine their effect on the rumen fermentation system. Materials and Methods: The first experiment was conducted using a randomized block design to select 14 strains of L. plantarum isolated from rumen cattle. The second experiment was arranged in a completely randomized design using two selected L. plantarum strains to determine their effects as a probiotic on rumen fermentation. The substrates used for in vitro fermentation were napier grass (Pennisetum purpureum) and concentrate in a 70:30 ratio. Results: From experiment 1, L. plantarum U32 was selected, because it produced low methane/total gas (27.39%) and strain U40 was selected because it had the highest dry matter and organic matter rumen disappearance (56.45 and 56.44%). In experiment 2, the addition of L. plantarum U32 and U40 as probiotics increased propionic acid and decreased acetic production (p<0.05), which led to a lower A:P ratio (p<0.05). The total volatile fatty acid and in vitro digestibility were not affected by the addition of L. plantarum. Probiotic addition increased lactic acid bacteria and the protozoa population (p<0.05) from the rumen fluid compared to the control. The total rumen bacteria were not significantly changed by the treatments. Conclusion: The addition of L. plantarum strains U32 and U40 as probiotics had beneficial effects for rumen fermentation due to increased propionic acid and decreased methane production.