Several studies have implicated β2-microglobulin (B2M) in osteoarthritis (OA) pathology. Of the main constituents of synovial tissue, synovial fibroblasts and macrophages, the latter play a pivotal role in inflammation. Although several studies have investigated the effects of B2M on synovial fibroblasts, few have examined the impact on synovial macrophages. Here, we investigated the effect of B2M on the expression profiles of inflammatory cytokines and matrix metalloproteases (MMPs) in synovial macrophages. Synovial macrophages were isolated from the osteoarthritic synovium using an anti-CD14 anti- body and magnetic isolation system. Synovial macrophages were stimulated with B2M for 6 and 24 h. Following stimulation, cell surface marker (CD80, CD163, CD206), cytokine [interleukin (IL)-6, IL-8, tumor necrosis factor α (TNF-α)] and matrix metalloprotease (MMP; MMP-9 and MMP-13) genes were evaluated by real-time PCR. Additionally, cytokine concentrations in cell culture supernatant were determined using enzyme-linked immunosorbent assay (ELISA). B2M significantly increased CD80 and decreased CD163 expression. In addition, B2M stimulation increased inflammatory cytokines at both the mRNA and protein levels. While B2M likewise elevated MMP-13 levels, there was no difference in MMP-9 expression between vehicle and B2M-treated cells. B2M increased M1 macrophage marker, inflammatory cytokine, and MMP-13 expression in synovial macrophages. B2M-related activation of synovial macrophages may thus be associated with OA pathology.