Abstract B lymphocyte-induced maturation protein 1 (Blimp1), encoded by the PRDM1 gene, is a multifunctional transcriptional regulator that has established roles in mouse T cells. However, the mechanisms underlying its function in the proliferation, suppressive function, and homeostasis of human regulatory T cells (Tregs) are not known. To directly evaluate the role of Blimp1 in human Tregs, Blimp1α, the active isoform of Blimp1 that has dominant expression, was knocked out by Crispr-Cas9 using dual-targeting sgRNAs. High knockout efficiency was verified at DNA, RNA, and protein level by T7E1, RT-PCR, and Western blot analyses, respectively. Knockout of Blimp1α in cultured human Tregs resulted in increased cell number, upregulated Ki67 expression, and enhanced [3H]thymidine incorporation, consistent with a normal inhibitory role of Blimp1α in the proliferation of human Tregs. Compared with control cells, Blimp1α-knockout Tregs showed high CD25 expression and enhanced tyrosine phosphorylation of STAT5 (pSTAT5), suggesting a potential role of Blimp1 in regulation of IL-2R signaling. In vitro suppression assay showed that knockout of Blimp1α slightly decreased the suppressive function of Tregs. Knockout of Blimp1α also increased the expressions of Tigit and CD62L, but decreased the expressions of Foxp3, CD73, and Bcl-2, suggesting that Blimp1α may contribute to maintaining Treg identity. Collectively, our results are consistent with a model whereby Blimp1α regulates homeostasis of human Tregs by constraining proliferation in part by limiting IL-2R signaling while promoting Treg function and identity.