Forkhead box P3 (FOXP3) is a transcription factor that is identified via CRISPR dissection. FOXP3 activity is an indicator of regulatory T cell (Treg) identity [1]. Tregs play essential role in maintaining the immunosuppressive landscape of tumor microenvironment (TME) [2]. FOXP3 controls several aspects of Treg differentiation and activity, and its deficiency accounts for severe autoimmune diseases in both human and mice [3]. FOXP3 expression in Tregs is determined by a combination of enhancers, transcription agents and epigenetic marks [4]. Loss of FOXP3 mediates Treg instability. The unstable cells acquire a pro-inflammatory phenotype and promote autoimmunity, but invigorating immune responses against cancer [5]. A longer (FOXP3 FL) and a shorter (FOXP3 ΔE2) isoform are two isoforms of FOXP3 identified in human. Solo expression of FOXP3 ΔE2 is representative of Treg instability, and patients with such motif are prone to develop autoimmunity [6]. FOXP3KO using CRISPR-Cas9 is an appropriate strategy for surveying Treg function [7]. The aim of this review is to give a comprehensive overview about the implication of FOXP3 in Tregs and its target in cancer immunotherapy based on current understanding.