<p>Thyroid cancer, the most prevalent endocrine malignancy, frequently presents with lymph node metastasis (LNM), a critical prognostic factor in influencing recurrence and survival. Even with progress in diagnostic and therapeutic strategies, the mechanisms underlying LNM, especially the role of microbial dysbiosis and metabolic reprogramming, remain incompletely understood. This study aimed to elucidate the interplay between gut microbiota, host metabolism, and LNM in thyroid cancer through a multi-tiered analytical framework to address this gap. We selected 90 patients with differentiated thyroid carcinoma (DTC) who underwent surgery. Before iodine-131 treatment, fecal samples were collected, and microbial diversity and metabolite profiles were analyzed using 16&#xa0;S rRNA sequencing and LC-MS-based metabolomics. Patients were stratified based on lymph node metastasis status (LNM vs. no metastasis), metastatic site (central vs. lateral neck metastasis), and metastatic burden (≤ 5, 5–10, and &gt; 10 metastatic nodes). Our findings revealed notable alterations in the composition of gut microbiota and metabolite profiles associated with LNM. At the phylum level, Proteobacteria was elevated in LNM + patients, while Bacteroidota and Fusobacteriota were depleted. At the genus level, Faecalibacterium and Escherichia_Shigella were enriched in LNM + patients, while Bacteroides was depleted. Metabolomics analysis identified several potential biomarkers, including osmanthuside A, niacin, and alpha-tocopherol, which could distinguish patients with and without LNM. Additionally, specific metabolites such as leukotriene-F4 and 3-Methoxytyramine showed marked differences between central and lateral neck metastasis groups. Our study underscores the potential influence of gut microbiota and metabolites on the advancement and metastasis of thyroid cancer progression. These findings offer a foundation for developing non-invasive biomarkers and therapeutic strategies targeting the gut-thyroid axis.</p>