Macrophages are dynamic cells whose phenotypes and functions are regulated by surrounding inflammatory mediators after pathogenic infection. Imbalanced polarization of classically activated (M1) and alternatively activated (M2) macrophages is closely associated with infection-related complications and their severity. The pathway of T-cell immunoglobulin mucin 3 (Tim-3)/galectin-9 (Gal-9) plays an important role in infection by regulating macrophage function. However, the effects of Tim-3/Gal-9 signalling on M1/M2 macrophage polarization are unclear. Bone marrow-derived macrophages (BMDMs) were stimulated with 0.1?μg/mL lipopolysaccharide (LPS). M1/M2 phenotypic macrophage markers were measured 0, 1, 3, 6, 12, and 24?h after stimulation, α-lactose was used to inhibit Gal-9, anti-mouse Tim-3 antibody was used to block Tim-3, recombinant mouse-Gal-9 (rm-Gal-9) was used to activate Tim-3, which were aimed to verify the role of the Tim-3/Gal-9 pathway in the balance of M1/M2 macrophages when stimulated with LPS. Short-term LPS stimulation upregulated Gal-9 expression and secretion, enhanced the association between Gal-9 and Tim-3, and activated the Tim-3/Gal-9 signalling pathway, eventually inhibiting M1 polarization. Long-term stimulation downregulated Gal-9 expression and secretion, reduced the association between Gal-9 and Tim-3, and inhibited the Tim-3/Gal-9 signalling pathway, eventually promoting M1 polarization, however, decreased M2 polarization and Gal-9 autocrine functions. Overall, LPS had a biphasic effect on BMDMs polarization through the Tim-3/Gal-9 pathway, which was time-dependent.