We had previously reported that Acanthamoeba castellanii (ACA) contains a mimicry epitope for proteolipid protein 139–151 capable of inducing central nervous system (CNS) autoimmunity in SJL/J mice. We now present evidence that ACA also contains a mimicry epitope for myelin basic protein (MBP) 89–101, a derivative from amoebic nicotinamide adenine dinucleotide dehydrogenase subunit 2 (NAD). The epitope, NAD 108–120, contains a discontinuous stretch of six amino acids in the core region (VVFFKNIILIGFL) sharing 46% identity with MBP 89–101 (VHFFKNIVTPRTP; identical residues are underlined). SJL mice immunized with NAD 108–120 develop encephalomyelitis similar to the disease induced by the cognate peptide. We demonstrate that NAD 108–120 induces T cells that cross-react with MBP 89–101; the antigen-sensitized T cells, which produce predominantly T helper (Th) 1 and Th17 cytokines, transfer disease in naive SJL recipients reminiscent of the disease induced with MBP 89–101. This is the first report to demonstrate that a solitary microbe can induce CNS autoimmunity by generating cross-reactive T cells for multiple myelin antigens.