Lyme disease is an infection caused by a tick-borne spirochete, Borrelia burgdorferi. Matrix metalloproteinase 9 (MMP-9) was selectively upregulated in the erythema migrans skin lesions of patients with acute Lyme disease. In this study, the mechanism of upregulation of MMP-9 was investigated in vitro and in vivo. The concentrations of MMP-9 and soluble CD14 were markedly elevated in serum from patients with acute Lyme disease and were also upregulated in U937 cells by B. burgdorferi in a time- and concentration-dependent manner. MMP-9 mRNA was expressed at baseline in fibroblasts in the presence or absence of B. burgdorferi. However, when fibroblasts were incubated with supernatants from U937 cells with B. burgdorferi or recombinant CD14, the expression of MMP-9 was significantly increased. This effect was completely abolished by the anti-CD14 antibody. These data suggest that the upregulation of MMP-9 by B. burgdorferi involves the CD14 pathway in infiltrating inflammatory cells. Fibroblasts could be recruited to amplify local production of MMP-9 by acquiring CD14 from macrophages.