ABSTRACT: Sarcopenia is a condition in which there is a loss of muscle caused by aging and it is one of the most significant factors that affects physical fragility. In recent years, the role of the gut-muscle axis has garnered attention as, along with the gut microbiota, it potentially plays a significant role in muscle regeneration, in addition to nutritional supplements and exercise training. Past studies have found that supplementation with Lactobacillus plantarum TWK10 could effectively increase the muscle mass of animals or adult humans. Therefore, in this study, we investigated whether the supplementation of L. plantarum TWK10 produces increased muscle mass and improves the functional performance of elderly persons with mild fragility. A total of 68 elderly subjects were recruited, of which 13 subjects were excluded or withdrew from the study. We adopted a double-blind design, and the 55 subjects were randomly divided into three groups: the placebo group, the TWK10 low-dose group (2 × 1010 CFU/day) (TWK10-L), and the TWK10 high-dose group (6 × 1010 colony-forming unit (CFU)/day) (TWK10-H). For 18 weeks, all subjects were required to regularly take experimental samples, perform functional activity testing, and have their body composition analyzed before the study and every six weeks after the intervention. Finally, 17 subjects in the placebo group, 12 subjects in the TWK10-L group, and 13 subjects in the TWK10-H group finished the study. It was found that supplementation with TWK10 had a tendency to increase and improve muscle mass, left hand grip strength, lower limb muscle strength, and gait speed and balance after the sixth week, especially in the TWK10-H group, and, as the supplement time was longer up to the 18th week, it had an even greater effect (p < 0.05). In conclusion, consecutive supplementation of L. plantarum TWK10 for more than six weeks could effectively improve the muscle strength and endurance of the elderly, reducing sarcopenia and physical fragility. This trial was registered as NCT04893746.