Type 1 diabetes (T1D) is an autoimmune disease mediated by T cells that attack the insulin-producing beta cells of the pancreas. Currently, there is no effective immunotherapy on the market that would reverse the progression of the disease. Yerba mate (YM) or Ilex paraguariensis is a natural herbal supplement that has been shown to have anti-inflammatory and hypoglycemic properties. This study aimed to examine the effects of YM in delaying the onset of T1D in the context of T lymphocytes in multiple low dose streptozotocin (MLD-STZ)-induced experimental C57BL/6 mouse model. Concentrations of 4% and 2% YM (w/v) were used ad libitum for 6 weeks. Administration of YM significantly reduced the incidence of T1D and hyperglycemia, while increasing the body weight of treated mice. Also, a trend of decreasing the incidence of T1D and decreasing the blood glucose level, but with a decrease in body weight, was demonstrated by the application of 4% YM in a pilot experiment on a model of spontaneously developing T1D non-obese diabetic NOD mice. Ex vivo analysis of splenic Tcells isolated from MLDSTZ-C57BL/6 mice showed that YM administered at both concentrations did not compromise cell viability, with a significant reduction in T-cell proliferation, transient changes in T-cell immunophenotypes (only with 2b% YM treatment) and reduction of the production of pro-inflammatory and anti-inflammatory cytokines, especially in the 4% YM treatment. T-cells directly exposed to YM in vitro showed a dose-dependent decrease in proliferation, an increased production of IL-2 with a decrease of IFN-λ. The obtained results suggest the preventative potential of YM in the experimental T1D and are consistent with other studies, confirming the hypoglycemic and immunomodulatory effects of YM on T-cells.