AbstractCD4⁺ CD25⁺ Foxp3⁺ Tregs have been shown to play a central role in immune homeostasis while preventing from fatal inflammatory responses, while Th17 cells have traditionally been recognized as pro-inflammatory mediators implicated in a myriad of diseases. Studies have shown the potential of Tregs to convert into Th17 cells and Th17 cells into Tregs. Increasing evidence have pointed out CD25 as a key molecule during this transdifferentiation process, however molecules that allow such development remain unknown. Here, we investigated the impact of NAD⁺ on the fate of CD4⁺ CD25⁺ Foxp3⁺ Tregs in-depth, dissected their transcriptional signature profile and explored mechanisms underlying their conversion into IL-17A producing cells. Our results demonstrate that NAD⁺ promotes Treg conversion into Th17 cells in vitro and in vivo via CD25 cell surface marker. Despite the reduced number of Tregs, known to promote homeostasis and an increased number of pro-inflammatory Th17 cells, NAD⁺ was able to promote an impressive allograft survival through a robust systemic IL-10 production that was CD4⁺ CD25⁺ Foxp3⁺ independent. Collectively, our study unravels a novel immunoregulatory mechanism of NAD⁺ that regulates Tregs fate while promoting allograft survival that may have clinical applications in alloimmunity and in a wide spectrum of inflammatory conditions.