Background: This study examined the impact of using Lactobacillus rhamnosus FERM P-15120 for the generation of bioactive peptides with antihypertensive action from fermented beef sausage. Methods: The Lactobacillus strain was used for meat fermentation at 35°C for up to 72 hours, and Angiotensin-converting enzyme (ACE)-inhibiting substances were measured to be released during fermentation. Fermented beef sausage extracts were purified and fractionated using OASIS® HLB 35cc (6g) LP extraction cartridge. Further purification and identification of peptide sequences were done using reversed-phased high-performance liquid chromatography (RP-HPLC) and protein sequencer. Results: A total of 16 peptide sequences from the active fractions were found to be 100% similar to various bovine skeletal muscle proteins. The molecular weights (MW) of all investigated peptides ranged from 423 to 2456, and the lengths of their amino acid sequences ranged from 3 to 23 amino acids. The IC₅₀ of peptides with high MW weight and ACE inhibitory activity was determined. NRYWH was discovered to be the most effective peptide, with an IC₅₀ value of 0.04 M. When compared to synthetic antihypertensive drugs, this peptide demonstrated good in vivo activity (captopril) due to it reduced systolic blood pressure by -32.76 ± 3.99 mm Hg (p < 0.05) in rats with spontaneous hypertension after 8 hours medication. Conclusion: The findings of this study suggest that naturally occurring peptides in fermented sausage may have therapeutic potential for human health.