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This study aimed to understand the genetic and metabolic traits of a <i>Lactiplantibacillus plantarum</i> JS21 strain and its probiotic abilities through laboratory tests and computer analysis. <i>L. plantarum</i> JS21 was isolated from a traditional fermented food known as “Jiangshui” in Hanzhong city. In this research, the complete genetic makeup of JS21 was determined using Illumina and PacBio technologies. The JS21 genome consisted of a 3.423 Mb circular chromosome and five plasmids. It was found to contain 3023 protein-coding genes, 16 tRNA genes, 64 rRNA operons, 40 non-coding RNA genes, 264 pseudogenes, and six CRISPR array regions. The GC content of the genome was 44.53%. Additionally, the genome harbored three complete prophages. The evolutionary relationship and the genome collinearity of JS21 were compared with other <i>L. plantarum</i> strains. The resistance genes identified in JS21 were inherent. Enzyme genes involved in the Embden–Meyerhof–Parnas (EMP) and phosphoketolase (PK) pathways were detected, indicating potential for facultative heterofermentative pathways. JS21 possessed bacteriocins <i>plnE</i>/<i>plnF</i> genes and genes for polyketide and terpenoid assembly, possibly contributing to its antibacterial properties against <i>Escherichia coli</i> (ATCC 25922), <i>Escherichia coli</i> (K88), <i>Staphylococcus aureus</i> (CMCC 26003), and <i>Listeria monocytogenes</i> (CICC 21635). Furthermore, JS21 carried genes for Na⁺/H⁺ antiporters, F₀F₁ ATPase, and other stress resistance genes, which may account for its ability to withstand simulated conditions of the human gastrointestinal tract <i>in vitro</i>. The high hydrophobicity of its cell surface suggested the potential for intestinal colonization. Overall, <i>L. plantarum</i> JS21 exhibited probiotic traits as evidenced by laboratory experiments and computational analysis, suggesting its suitability as a dietary supplement.