Traditional fermented foods harbor functionally diverse microbial communities that represent an underexplored source of probiotic strains. Lactic acid bacteria were systematically isolated from traditionally fermented bamboo shoots collected across Zhejiang Province, China, with the aim of identifying superior probiotic candidates. Among the isolates recovered, Lactococcus lactis ZB2 demonstrated robust gastrointestinal tolerance, favorable adhesion properties, and broad-spectrum antimicrobial activity, consistently matching or exceeding the reference strain Lactobacillus rhamnosus GG across all evaluated in vitro criteria. Molecular identification via 16S rRNA gene sequencing confirmed ZB2 as L. lactis subsp. lactis (>99.7% sequence identity). To evaluate its in vivo effects, ZB2 (1 × 109 CFU/day, 200 μL of bacterial suspension in 0.01 M PBS, pH 7.4) was administered by oral gavage to healthy C57BL/6 J mice for 28 days. ZB2 supplementation significantly reinforced intestinal epithelial barrier integrity, evidenced by reduced serum permeability markers (FITC-dextran flux, DAO, D-lactic acid, and LPS), upregulation of tight junction proteins (ZO-1, Occludin, Claudin-1) and the mucin gene Muc2, and a significant increase in goblet cell number per crypt. Immune homeostasis was modulated toward an anti-inflammatory phenotype, characterized by elevated serum IL-10 and TGF-β, reduced IFN-γ, downregulation of colonic pro-inflammatory cytokines (Tnf-α, Il-6, Il-1β), and upregulation of the antimicrobial peptides Reg3γ and β-defensin 1. 16S rRNA amplicon sequencing revealed selective enrichment of butyrate-producing genera within the Lachnospiraceae family, accompanied by marked increases in fecal short-chain fatty acid concentrations—particularly butyrate and propionate. Systemic antioxidant capacity was also enhanced, as reflected by elevated SOD, CAT, and GSH-Px activities and reduced malondialdehyde. These findings support the characterization of L. lactis ZB2 as a multifunctional probiotic candidate and highlight Zhejiang fermented bamboo shoots as a valuable, underexplored reservoir of superior probiotic strains with broader functional food development potential.