Objective To study the effect of deletion of Toll-like receptor 2 (TLR2) gene on insulin resistance and polarization of macrophages in mice. Methods The wild-type (WT) and TLR2 knockout (TLR2-/-) C57BL/6 male mice, aged 28 days, were selected, with 12 mice in each group. The genotype of each mouse was identified by PCR. After mice were fed with basic diet for 3 months, the glucose tolerance test (GTT) and insulin resistance test (ITT) were performed. The mononuclear cells isolated from peripheral blood were stimulated with GM-CSF/IFN-γ and M-CSF/IL-4/IL-13, respectively, to induce differentiation to M1-like and M2-like macrophages. The CD11b, F4/80, CD11c, CD206 and early growth response 2 (EGR2) were detected by flow cytometry to determine the phenotype of macrophages. The levels of TNF-α, IL-6 and IL-10 in the culture supernatant of macrophages were detected using ELISA. Results The result of PCR identification was consistent with the genotype of mice. Compared to WT mice, TLR2-/- mice exhibited the significantly improved glycemic control at 30 min during GTT and the significantly increased insulin sensitivity at 15 minutes during ITT. The flow cytometry showed that M1 markers decreased and M2 macrophages increased in the TLR2-/- mice. ELISA showed that the levels of IL-6 and TNF-α significantly decreased in the culture supernatant of M1 macrophages, while the level of IL-10 significantly increased in the culture supernatant of M2 macrophages in TLR2-/- mice compared with WT mice. Conclusion TLR2 signal has an effect on the polarization of macrophages and makes macrophages tend to switch to M1 phenotype. A higher number of pro-inflammatory factors secreted by M1 macrophages contribute to a low-grade inflammation state in the body, which leads to a decrease in glucose tolerance and insulin sensitivity.