In sepsis, immunosuppression is commonly observed as lipopolysaccharide (LPS) tolerance in macrophages. Leukocyte immunoglobulin-like receptor B2 (LILRB2) is an inhibitory receptor on immune cells that may play a crucial role in the immunosuppressive phenotype of LPS-tolerant macrophages, although its exact function in sepsis remains unclear. In this study, macrophages were exposed to single or sequential LPS doses to induce LPS stimulation or tolerance. Cell viability was assessed using CCK-8 assay, apoptosis, and macrophage polarization were detected by flow cytometry, and pro-inflammatory cytokine levels were measured by RT-qPCR and ELISA. Molecular interactions were explored using Co-IP, ChIP, and dual-luciferase assays, while mRNA and protein expression were assessed by RT-qPCR and Western blotting. The results showed that LILRB2 was upregulated in macrophages following LPS stimulation, with a more significant increase in the LPS-tolerant group. Knocking down LILRB2 reversed the immunosuppressive phenotype of LPS-tolerant macrophages and restored the inhibition of MyD88/NF-κB signaling and p65 nuclear translocation caused by LPS tolerance. Mechanistically, LILRB2 interacted with Toll-like receptor 8 (TLR8) to inhibit the MyD88/NF-κB signaling pathway in LPS-tolerant macrophages. Furthermore, the upregulation of the Spi-1 proto-oncogene (SPI1) enhanced the immunosuppressive phenotype by transcriptionally activating LILRB2. In conclusion, SPI1 upregulation promoted the immunosuppressive phenotype of LPS-tolerant macrophages by activating LILRB2 transcription, which inhibited TLR8-mediated MyD88/NF-κB signaling. This study clarifies the role of LILRB2 and its underlying mechanisms in LPS-tolerant macrophages.