CD4 + Foxp3 + regulatory T (Treg) cells are key players in keeping excessive inflammation in check. Mounting evidence has shown that Treg cells exert much more diverse functions in both immunological and non-immunological processes. The development, maintenance and functional specification of Treg cells are regulated by multilayered factors, including antigens and TCR signaling, cytokines, epigenetic modifiers and transcription factors (TFs). In the review, we will focus on TFs by summarizing their unique and redundant roles in Treg cells under physiological and pathophysiological conditions. We will also discuss the recent advances of Treg trajectories between lymphoid organs and non-lymphoid tissues. This review will provide an updated view of the newly identified TFs and new functions of known TFs in Treg biology.