Abstract Forkhead Box P3+ (FOXP3+) T cells are regulatory cells important for maintaining immune tolerance. While chemokine- and other homing-receptors are important for T cell migration, it has been unclear how they are regulated in FOXP3+ T cells. We thoroughly investigated, ex vivo and in vitro, the regulation of chemokine receptor expression on human FOXP3+ T cells in neonatal cord blood, adult peripheral blood, and tonsils. We found that human FOXP3+ T cells undergo changes in trafficking receptors according to their stages of activation and differentiation. FOXP3+ T cells are divided into CD45RA+ (naive type) and CD45RO+ (memory type) FOXP3+ T cells in neonatal blood, adult blood, and tonsils. CD45RA+FOXP3+ T cells mainly express lymphoid tissue homing receptors (CD62L, CCR7, and CXCR4), while CD45RO+FOXP3+ T cells highly express both Th1 and Th2-associated trafficking receptors along with the lymphoid tissue homing receptors at reduced frequencies. Up-regulation of Th1/Th2-associated trafficking receptors begins with activation of CD45RA+FOXP3+ T cells and is completed after their differentiation to CD45RO+ cells. Some chemokine receptors such as CXCR5 and CXCR6 are preferentially expressed by many FOXP3+ cells at a specific stage (CD69+CD45RO+) in tonsils. Our in vitro differentiation study demonstrated that CD45RA+FOXP3+ T cells indeed undergo chemokine receptor switch from CD45RA+ (secondary lymphoid tissue homing) to CD45RO+ type (lymphoid and nonlymphoid tissue homing). The orderly regulation of trafficking receptors in FOXP3+ T cells according to stages of differentiation and activation is potentially important for their tissue-specific migration and regulation of immune responses in humans.