〔Abstract〕 Objective To investigate the effects of Bruton's tyrosine kinases(Btk) inhibitor, ibrutinib, on inflammatory response in high glucose-induced macrophages and to discover the underlying mechanisms. Methods In this study, bone marrow-derived macrophages(BMMs) were used. After the optimization of the experimental conditions, the BMMs were divided into four groups: low-glucose group(LG), low-glucose+ibrutinib control group(LG+PCI-32765), high-glucose group(HG), and high-glucose+ibrutinib group(HG+PCI-32765). The macrophages were detected by flow cytometry. The expression of inflammatory cytokines in cell and in cell supernatant, including the monocyte chemo-attractant protein-1(MCP-1), the tumor necrosis factor-alpha(TNF-α) and the interleukin-1 beta(IL-1β) and the mRNA, were detected by enzyme-linked immuno sorbent assay and quantitative real-time PCR. The iNOS, Btk, ERK, JNK, p38 MAPK, NF-κB p65, IκB and the corresponding phosphorylated proteins were detected by Western blot. The activation of macrophages and nuclear transfer of p65 were detected by laser confocal microscopy. Results Compared with the LG group, the expression of inflammatory cytokines in the HG group increased(P<0.05), while that decreased in the HG+PCI-32765 group compared with the HG group(P<0.05). The expression of p-Btk was higher in the HG group(P<0.01), and the p-ERK, p-JNK, p-p38 MAPK, NF-κB p-p65 and p-IκB in the HG group were also higher(P<0.05) compared with the LG group, and the Btk inhibitor, PCI-32765, could reduce the expression of p-Btk, p-ERK, NF-κB p-p65 and p-IκB(P<0.05). The macrophages were activated by high-glucose, and the expression of iNOS increased(P<0.01), while PCI-32765 could reduce its expression(P<0.05). By using laser confocal microscopy, it was found that under high-glucose stimulation, p65 had nuclear-transfer, and PCI-32765 could reduced it. Conclusion Ibrutinib is able to inhibit the high glucose-induced activation of macrophages and suppress the release of inflammatory cytokines by down-regulating the ERK and the NF-κB signaling pathways.