Hashimoto’s thyroiditis (HT) is one of the most common organ-specific autoimmune diseases, characterized by chronic thyroid gland inflammation. Helper T (Th) cells, whose surface receptors are highly glycosylated, are involved in the pathomechanism of HT. Our study aimed to characterize N-glycosylation profiles in two pools of T cells, defined by the expression of late activation marker () and CD25-negative cells () in HT. Two study groups were recruited: HT1 with elevated thyroid autoantibodies and TSH level within the normal range without hypothyroidism, and HT2, hypothyroid HT patients, adequately metabolically controlled while on L-thyroxine replacement therapy, and healthy subjects to the control group (CTR). N-glycans from cell proteins, released using N-glycosidase F, were analyzed by MALDI-Tof mass spectrometry. RT-qPCR was used to determine the expression of selected glycogenes. We found significant differences in the glycome of and cells. In homeostasis (CTR), a predominance of complex-type glycans was observed in cells, whereas the oligomannose-type structures prevail in lymphocytes. In autoimmunity and progressive thyroid dysfunction, the rearrangement of N-glycans in Th cells was observed, in opposite directions in the pools. Complex-type structures are replaced by oligomannose forms in in the HT1 group, while in HT2, a restoration of glycosylation profile to the level of CTR was detected. cells accelerated complex-type synthesis in HT1, which was normalized in HT2 patients. Changes in the profile of N-linked glycans are partially reflected in the expression of mannosidases and glycosyltransferases. Our study demonstrates for the first time the diverse N-glycosylation profiles in and cells, and the rearrangement of N-glycan structures specific for each pool of Th cells in HT. Further studies are needed to determine the functional aspect of the identified N-glycosylation changes during thyroid autoimmunity.