Regulatory T cells (Tregs), which express CD4 and FOXP3, are critical for modulating the immune response and promoting immune tolerance. Consequently, methods to expand Tregs for therapeutic use are of great interest. While transfer of Tregs after massive ex vivo expansion can be achieved, in vivo expansion of Tregs would be more practical. Here, we demonstrate that targeting the CD45 tyrosine phosphatase with a tolerogenic anti-CD45RB mAb acutely increases Treg numbers in WT mice, even in absence of exogenous antigen. Treg expansion occurred through substantial augmentation of homeostatic proliferation in the preexisting Treg population. Moreover, anti-CD45RB specifically increased Treg proliferation in response to cognate antigen. Compared with conventional T cells, Tregs differentially regulate their conjugation with DCs. Therefore, we determined whether CD45 ligation could alter interactions between Tregs and DCs. Live imaging showed that CD45 ligation specifically reduced Treg motility in an integrin-dependent manner, resulting in enhanced interactions between Tregs and DCs in vivo. Increased conjugate formation, in turn, augmented nuclear translocation of nuclear factor of activated T cells (NFAT) and Treg proliferation. Together, these results demonstrate that Treg peripheral homeostasis can be specifically modulated in vivo to promote Treg expansion and tolerance by increasing conjugation between Tregs and DCs