Abstract: Background and Objective: This study aimed to achieve optimal microbial protein synthesis by adding various levels of gambier leaf residue (GLR) to cattle feed supplement (CFS). Materials and Methods: Gambier (Uncaria gambir Roxb) leaf residue containing 9.96% condensed tannins was added at levels of 0, 2.5, 5.0 and 7.5% (DM) to CFS formula containing 29% crude protein and then tested in vitro rumen digestion. Results: Addition of 5% GLR significantly decreased the rumen protozoan population by 24.43% (p<0.05) and decreased the NH₃-N content (p<0.01) but did not affect the pH or total volatile fatty acid (VFA) content (p>0.05). The mean of the microbial biomass was 111-285 mg 100^–1 mL of rumen fluid and the microbial protein concentration was 59-157 mg 100^–1 mL of rumen fluid, which was highest at 5.0 GLR (p<0.01). Overall, the rate of microbial biomass production, rate of microbial protein synthesis and efficiency of microbial protein synthesis were 7.30-10.18 mg 100^–1 mL h^–1, 3.86-5.62 mg 100^–1 mL h^–1 and 16-47 mg microbial protein 100^–1 mg digested organic matter, respectively, with the highest rate at 5.0% GLR and the lowest rate in controls (p<0.01). Conclusion: Addition of 5.0% GLR to CFS can optimize microbial protein synthesis in the rumen.