Objective. Follicular regulatory T (Tfr) cells act as the regulatory counterpart of follicular helper T (Tfh) cells to suppress germinal center (GC) B cell differentiation. We recently showed that interleukin-21 (IL-21) promoted Tfh cell differentiation in autoimmune BXD2 mice that develop spontaneous GCs. This study was undertaken to determine the modulatory effects of IL-21 on Tfr cells and the Tfr cell to Tfh cell balance in BXD2 mice. Methods. The percentage and phenotype of Tfr cells were determined in BXD2 and BXD2-IL21-/- mice. The effects of IL-21 on Tfr cells and the Tfr cell:Tfh cell ratio were evaluated. Sorted Tfr cells from BXD2-IL21-/- mice were cocultured with Tfh cells and B cells, or transferred into BXD2 mice to determine their function. Results. The percentages and numbers of GC B cells and Tfh cells were significantly reduced, but the percentage of Tfr cells was 2-fold higher in BXD2-IL21-/- mice than in wild-type BXD2 mice. Administration of AdIL-21 to BXD2-IL21-/- mice decreased the percentages and numbers of Tfr cells and the Tfr cell:Tfh cell ratio but increased the number of GC B cells in the spleen. Recombinant murine IL-21 suppressed FoxP3 and significantly reduced Tgfb1, Il2, and Gitr but enhanced Il21, Il6, Pd1, Cxcr5, and Icos expression in Tfr cells. IL-21 also counteracted Tfr cell-mediated inhibition of antibody secretion in the Tfh cell-B cell coculture system. Transfer of Tfr cells into young BXD2 mice reduced GC size and decreased the numbers of autoantibody-producing B cells. Conclusion. Our findings indicate that high levels of IL-21 selectively enhance Tfh cell differentiation but inhibit Tfr cell commitment and the suppressive function of Tfr cells on Tfh cells and B cells, suggesting that IL-21 skews the balance from Tfr cells to Tfh cells to promote autoreactive GC reactions in BXD2 mice. © 2014, American College of Rheumatology.