Regulatory T cell (Treg) therapy has shown therapeutic potential for treating inflammatory bowel disease (IBD). Animal studies have also shown that intestinal antigen-specific Tregs are more potent at inhibiting colitis than polyclonal Tregs. Chimeric antigen receptor (CAR) technology offers a novel approach to generate Tregs recognising IBD-associated antigens in an MHC-independent manner. We optimised several expansion methods for ex vivo isolated mouse polyclonal and in vitro generated CAR Tregs for preclinical applications. We compared Tregs sorted as Foxp3eGFP+ cells versus CD25+Foxp3eGFP+ cells, finding that there were no significant differences in phenotype or function, but that the latter method was optimal in terms of yield and expansion potential. We also compared expansion in the presence/absence of the mTORC1 inhibitor rapamycin, finding that inclusion of rapamycin during expansion resulted in optimal preservation of the expected Treg phenotype, with high Foxp3 expression, low production of inflammatory cytokines and preserved suppressive function. Mouse Tregs transduced with a CAR-encoding retrovirus and expanded in vitro using this optimized method maintained their phenotype and suppressive capacity. In vivo, adoptive transfer of expanded untransduced or CAR-transduced Tregs protected mice from colitis in a T cell transfer model. Remarkably, a Treg-mediated suppressive effect was observed even at a 1:100 ratio (Treg:T effector cell), suggesting that in vitro expanded Tregs have a heightened suppressive function compared to ex vivo cells. These optimized methods can now be applied to test the biological effects of CAR-Tregs in a variety of pre-clinical models of disease.