Probiotics play a crucial role in promoting host health by modulating the composition of the gut microbiota through the production of their own bioactive metabolites. The aim of the study was to investigate the anti-atrophic effects of Lacticaseibacillus paracasei EFEL6501 (EFEL6501) in dexamethasone (DEX)-treated C2C12 myotubes and a mouse model. In vitro experiments demonstrated that specific bioactive metabolites present in the cell culture supernatant (CS) and lysate supernatant (LS) of EFEL6501 alleviated muscle degradation and restored muscle protein synthesis in DEX-induced C2C12 myotubes. Similarly, EFEL6501 supplementation in mice significantly enhanced muscle thickness (6.09 mm), grip strength (117.87 g), and the cross-sectional area (CSA) (34.11 μm2) of the gastrocnemius muscle, compared to the DEX group (5.70 mm, 106.87 g and 29.79 μm2, respectively), by suppressing protein degradation pathways and improving muscle differentiation. Furthermore, EFEL6501 positively modulated the gut microbiota composition by increasing the abundance of beneficial bacteria, including Lactobacillus reuteri (7.19%), Bifidobacterium choerinum (25.66%), Bacteroides uniformis (0.29%), Allobaculum (0.63%), and Faecalibaculum (18.00%) compared to the DEX group (3.44%, 0.75%, 0.14%, -0.63%, and 8.53%, respectively), while also elevating acetate concentrations from 1.57 ± 0.27 mM to 1.97 ± 0.16 mM. Taken together, EFEL6501 may serve as a potential functional probiotic for preventing muscle atrophy by regulating muscle metabolism and gut microbiota composition.