Background <p>Evidence suggests that <i>Lactiplantibacillus plantarum</i> (<i>LP</i>) exerts a therapeutic effect in ulcerative colitis (UC). However, the temporal dynamics underlying the influence of <i>LP</i> AS21 on the efficacy of mesalazine (MSA) treatment in UC remain poorly defined. This study aims to elucidate the time-dependent impact of <i>LP</i> AS21 on MSA-mediated therapeutic outcomes in UC.</p> Methods <p>Colitis was induced in female C57BL/6J mice using 3% DSS for one week, followed by a three-week treatment phase with weekly evaluations. Disease progression was monitored via body weight, DAI, and colon length measurement. Histology, serum markers, and immunohistochemistry were used to assess inflammation, oxidative stress, and epithelial integrity at multiple time points. Subsequently, intestinal contents were collected for 16&#xa0;S rRNA gene sequencing and untargeted LC-MS metabolomics to track temporal microbial and metabolic dynamics. Correlation analyses were performed to assess the association of specific microbiota and metabolites with cytokines and antioxidant markers.</p> Results <p>Time-dependent <i>LP</i> AS21 intervention significantly modulated the therapeutic efficacy of MSA in DSS-induced colitis. <i>LP</i> treatment during the second and third weeks promoted recovery of body weight, improved DAI, and restored mucosal architecture compared with the first week. The temporal administration of <i>LP</i> modulated the host response to MSA treatment by enhancing antioxidant defenses, including catalase, superoxide dismutase, and glutathione, while suppressing pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β, and IFN-γ, with the most pronounced effects observed during the second and third weeks. Additionally, DSS administration increased microbial richness and promoted opportunistic pro-inflammatory taxa, including <i>Akkermansia</i> and Proteobacteria. Extended <i>LP</i> intervention influenced MSA efficacy by partial restoration of gut microbial diversity and enrichment of beneficial taxa. This microbial restoration was associated with normalization of the metabolic profile, especially in steroid biosynthesis and glycerophospholipid pathways. Key metabolites, including 5-aminopentanoic acid, GpCho (16:0/20:5), and 25-hydroxycholecalciferol, were significantly restored. These metabolite-driven changes in glycerophospholipid and steroid metabolism correlated with improved redox homeostasis and immune regulation, highlighting their potential as biomarkers to assess time-dependent therapeutic responses of <i>LP</i>-augmented MSA treatment in UC.</p> Conclusion <p><i>LP</i> AS21 intervention improves MSA efficacy in DSS-induced colitis by modulating immunometabolic networks and gut microbiota, with treatment duration critically shaping therapeutic outcomes.</p> Graphical abstract <p></p>