Background & Aims Kruppel-like Factor 14 (KLF14) proteins function as epigenetic reprogramming factors during cell differentiation in many cell populations and in engineered induced pluripotent stem cells. In this study, we determined the function of KLF14 in the regulation of forkhead box protein 3 (FOXP3), a transcription factor critical for T regulatory (Treg) cell differentiation. Methods We studied the effects of KLF14 on FOXP3 expression at the level of the protein and mRNA. We evaluated the functional relevance of KLF14 to FOXP3+ Treg cells in vitro and in vivo through suppression assays and two colitis models. Finally, we analyzed the effect of KLF14 on the epigenetic landscape of the FOXP3 promoter locus through chromatin immunoprecipitation (ChIP) assay. Results KLF14, induced upon activation of naïve CD4+ T cells, segregates to the FOXP3- population and is inversely associated with FOXP3 expression and Treg function. KLF14 knockout (KO) CD4+ cells differentiated into adaptive Tregs more readily in vitro and in vivo. KLF14 KO cells demonstrated an enhanced Treg suppressor function in vitro and in vivo. KLF14 repressed FOXP3 at the level of the mRNA and protein, and by ChIP assay KLF14 was found to bind to the Treg-specific demethylation region (TSDR) enhancer region of FOXP3. Furthermore, loss of KLF14 reduced the levels of H3K9me3, HP1, and Suv39H1at the TSDR. Conclusions These results outline a novel mechanism by which KLF14 regulates Treg cell differentiation via chromatin remodeling at the FOXP3 TSDR. To our knowledge, this is the first evidence to support a role for KLF14 in maintaining the differentiated state of Treg cells, with an outline of a potential mechanism to modify the expression of immune genes such as FOXP3 that are critical to T-cell fate.