Abstract Chemokines are key regulators of cell trafficking, and dipeptidyl peptidase IV/CD26 (CD26) inactivates chemokines. Here we show that the CD26-processed chemokines SDF1α/CXCL12 and RANTES/CCL5, in contrast to a control chemokine not processed by CD26, are potent inducers of cell surface expression of thrombospondin-1 (TSP-1) in T lymphocytes through a CD26-controlled mechanism and that TSP-1 stimulates expression of lipoprotein receptor related protein/CD91. Accordingly, intact TSP-1 and a peptide mimetic of a sequence in TSP-1 were sufficient to stimulate CD91 expression. The chemokine-induced expression of TSP-1 and CD91 was mimicked by inhibitors of CD26 and CXCL12 and CCL5 as well as inhibitors of CD26 stimulated polarized cytoplasmic spreading and migration through TSP-1. Silencing of CD26 using small interfering RNA or Ab-induced modulation of CD26 also increased TSP-1 expression and enhanced cytoplasmic spreading and T cell migration markedly. These results indicate that CD26 is an endogenous inhibitor of T cell motility through inhibition of TSP-1 expression and that chemokines stimulate cell polarity and migration through abrogation of the CD26-dependent inhibition. This suggests that T cell motility is regulated by a cascade of interacting cell surface molecules.