The fate of Foxp3+ regulatory T (Treg) cells responding during autoimmunity is not well defined. We observed a marked elevation in KLRG1+ (where KLRG1 stands for killer cell lectin-like receptor G1) CNS-infiltrating Treg cells in experimental autoimmune encephalomyelitis (EAE), and assessed their origin and properties. KLRG1+ Treg cells showed increased activation marker expression, Foxp3 and CD25 levels, and more rapid cell cycling than KLRG1− cells. KLRG1− Treg cells converted into KLRG1+ cells and this was increased in autoimmune inflammation. Conversion was unidirectional; KLRG1+ Treg cells did not revert to a KLRG1− state. KLRG1+ but notKLRG1−Treg cells survived poorly, indicative of terminal differentiation. This was associated with diminished BCL2 and increased apoptosis of isolated cells. KLRG1 was also upregulated on iTreg cells after transfer and EAE induction or on iTreg cells developing spontaneously during EAE. KLRG1+ Treg cells produced more IL-10 and had altered effector cytokine production compared with their KLRG1− counterparts. Despite their differences, KLRG1+ and KLRG1− Treg cells proved similarly potent in suppressing EAE. KLRG1+ and KLRG1− populations were phenotypically heterogeneous, with the extent and pattern of activation marker expression dependent both on cellular location and inflammation. Our results support an extensive diversification of Treg cells during EAE, and associate KLRG1 with altered Treg-cell function and senescence.