α-Galactosylceramide (α-GC) is widely known to activate invariant natural killer T (iNKT) cells to suppress myelin antigen-specific Th1 responses, protecting susceptible mice against experimental autoimmune encephalomyelitis (EAE). Here, we demonstrate an unexpected finding that high doses of α-GC exacerbated, rather than ameliorated, EAE. Similar results were observed when MOG 35-55-specific T cells treated with high-dose α-GC were transferred into naïve syngeneic recipient mice. Further study showed that high doses of α-GC directly enhance the Th17 and Th1 response by activation of CD4+ CD44+ memory T cells through phosphorylation of STAT3 and activation of NF-κB. Unlike the activation of iNKT cells by low doses of α-GC, high doses of α-GC directly interacted with CD1d expressed on T cells and activated Th17 and Th1 cells. Furthermore, antigen-presenting cells (APCs) predominantly express CD1d1, whereas the majority of CD4+ T cells express CD1d2. Knockdown of CD1d1 or CD1d2 gene expression by RNAi interfered with the activation of iNKT or Th17/Th1 cells, respectively. Therefore, α-GC treatment could improve or worsen EAE by engaging either APCs or Th17/Th1 cells depending on the dose used.