Borrelia burgdorferi is the etiologic agent of Lyme disease, a multisystem inflammatory disorder that principally targets the skin, joints, heart, and nervous system. The role of T lymphocytes in the development of chronic inflammation resulting from B. burgdorferi infection has been controversial. We previously showed that natural killer T (NKT) cells with an invariant (i) TCR α chain (iNKT cells) recognize glycolipids from B. burgdorferi, but did not establish an in vivo role for iNKT cells in Lyme disease pathogenesis. Here, we evaluate the importance of iNKT cells for host defense against these pathogenic spirochetes by using Vα14i NKT cell-deficient (Jα18^−/−) BALB/c mice. On tick inoculation with B. burgdorferi, Jα18^−/− mice exhibited more severe and prolonged arthritis as well as a reduced ability to clear spirochetes from infected tissues. Vα14i NKT cell deficiency also resulted in increased production of antibodies directed against both B. burgdorferi protein antigens and borrelial diacylglycerols; the latter finding demonstrates that anti-glycolipid antibody production does not require cognate help from Vα14i NKT cells. Vα14i NKT cells in infected wild-type mice expressed surface activation markers and produced IFNγ in vivo after infection, suggesting a participatory role for this unique population in cellular immunity. Our data are consistent with the hypothesis that the antigen-specific activation of Vα14i NKT cells is important for the prevention of persistent joint inflammation and spirochete clearance, and they counter the long-standing notion that humoral rather than cellular immunity is sufficient to facilitate Lyme disease resolution.