All-trans retinoic acid (ATRA) induces the expression of gut-homing receptors on T cells during activation in vitro. We investigated the effect of ATRA as an in vivo adjuvant during vaccination on the formation and migration of memory T cells in C57BL6 mice. ATRA treatment during priming with recombinant adenovirus expressing the LCMVgp33 epitope resulted in increased memory T cells in mucosal tissues and higher frequency of central memory T cells in spleen.  T cells primed in the context of ATRA exposure mounted stronger responses to systemic challenge with MVAgp33, and were more resistant to challenge with VVgp33 at vaginal surfaces. In co-transfer experiments, memory CD8 T cells from ATRA treated mice exhibited greater proliferation and increased gut homing following MVAgp33 challenge. Thus ATRA may be useful as an adjuvant during vaccination to enhance T cell responses at mucosal sites.