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We observed a remarkable reduction in the frequency and immunosuppressive activity of splenic CD4⁺CD25⁺ T cells in C57BL/6 mice with MOG_33–55‐induced experimental autoimmune encephalomyelitis (EAE). Our study revealed that pertussis toxin (PTx), one component of the immunogen used to induce murine EAE, was responsible for down‐regulating splenic CD4⁺CD25⁺ cells. Treatment of normal BALB/c mice with PTx in vivo reduced the frequency, suppressive activity and FoxP3 expression by splenic CD4⁺CD25⁺ T cells. However, PTx treatment did not alter the expression of characteristic phenotypic markers (CD45RB, CD103, GITR and CTLA‐4) and did not increase the expression of CD44 and CD69 by the residual splenic and lymph node CD4⁺CD25⁺ T cells. This property of PTx was attributable to its ADP‐ribosyltransferase activity. PTx did not inhibit suppressive activity of purified CD4⁺CD25⁺ T …