Pediococcus pentosaceus (P. pentosaceus) is a ubiquitous lactic acid bacterium recognized for enhancing the sensory properties, dietary benefits, and safety of fermented foods. This study isolates and characterizes a Tibetan kefir-derived strain, P. pentosaceus K6, using integrated genomic and phenotypic approaches. Whole-genome sequencing and in vitro assays confirmed a favorable safety profile for K6, showing no hemolytic activity or harmful metabolite production, and lacking pathogenic virulence determinants, transferable antibiotic resistance genes, or functional plasmid conjugation elements. K6 showed strain-specific probiotic features, including 83.83% survival at pH 3.0 for 2 h, 71.16% survival under simulated gastrointestinal digestion, 89.30% auto-aggregation at 48 h, and strong inhibition against Listeria monocytogenes (29.83 ± 0.06 mm). Compared with previously reported P. pentosaceus strains, K6 uniquely combines gastrointestinal tolerance, adhesion-related traits, and anti-Listeria activity. The antiSMASH analysis identified putative type III polyketide synthase and terpene gene clusters, providing genetic candidates for this antimicrobial phenotype. Furthermore, specific carbohydrate-active enzymes and exopolysaccharide biosynthetic genes indicate strong potential for carbohydrate metabolism and food matrix adaptation. Collectively, these genomic and phenotypic findings support P. pentosaceus K6 as a highly promising candidate for probiotic and biopreservation applications.