Background <p>Exploring the differential mechanism between Brucella spondylitis (BS) and Tuberculous spondylitis (TS) is crucial for the full treatment and management of this disease.</p> Methods <p>Spinal samples of 14 patients with BS, 13 patients with TS, and 13 controls were recruited. Among them, 4 BS patients, 3 TS patients, and 3 controls were randomly selected for high-throughput sequencing. Differential expression analysis was performed among the three groups to identify the regulatory relationships between lncRNAs and mRNAs. Weighted Gene Co-expression Network Analysis (WGCNA) and enrichment analysis were constructed for the differentially expressed mRNAs (DEmRs) and differentially expressed lncRNAs (DElncRs). Finally, experiments were conducted using the remaining samples for validation.</p> Results <p>A total of 213 DEmRs and 97 DElncRs were specific to BS, 242 DEmRs and 54 DElncRs were specific to TS. In the identified 16 co-expression modules, the black module showing the highest positive correlation with BS and the lightcyan module showing the highest positive correlation with TS. Enrichment analysis revealed that genes in the black module were primarily associated with the Toll-like receptor signaling pathway. Genes in the lightcyan module were mainly associated with Th1 and Th2 cell differentiation, as well as Th17 cell differentiation. Western blot confirmed increased TLR4 expression in BS, and flow cytometry showed elevated Th2 cells and reduced Th17 cells in TS.</p> Conclusion <p>CXCL8, CCL3, and CCL4L2 may be involved in the pathological process of BS through TLR4 signaling. Similarly, HLA-DRB3 and HLA-DRB1 may be involved in the pathological process of TS through the regulation of T-cell subgroups.</p>