CD133+ cancer stem cells are responsible for thyroid cancer initiation. The regulatory pathways essential for sustaining the self-renewal of thyroid cancer stem cells remain largely unknown. Glutamate signaling regulates the self-renewal ability of stem cells. In the present study, we found that the level of glutamate was higher in CD133+ thyroid cancer cells than in CD133- thyroid cancer cells. The transcriptional level of glutamate aspartate transporter SLC1A3 was high in CD133+ thyroid cancer cells. Activation of NF-KB signaling by CD133 was responsible for SLC1A3 high transcription level in CD133+ thyroid cancer cells. Knock down of SLC1A3 significantly reduced the level of glutamate and inhibited the self-renewal activity and tumorigenicity of CD133+ thyroid cancer cells. Overexpression of SLC1A3 rescued the negative effect of CD133 knockdown on the self-renewal capability of CD133+ thyroid cancer cells. Taken together, CD133 promotes the self-renewal capacity of CD133+ thyroid cancer cells at least partly through activation of SLC1A3 expression. (C) 2019 Elsevier Inc. All rights reserved.