Molecules that regulate leukocyte trafficking to sites of inflammation or homing to secondary lymphoid organs are crucial for an effective immune response. L-selectin is one important adhesion molecule required for these migration processes to occur. Tetraspanins are a family of integral membrane proteins, which can regulate cellular migration, principally through their regulation of integrins. CD53 is a leukocyte-specific tetraspanin, the function of which has only been investigated in vitro. Here, we present the first study of the phenotype of a novel CD53-/- mouse and show compelling evidence that CD53 regulates L-selectin. When compared to WT mice, splenic cellularity was normal in CD53-/- mice; however, peripheral lymph node (PLN) cellularity was markedly decreased with an 85% reduction in B cell number. We found that CD53 is essential for the expression of L-selectin on B cells and neutrophils, but interestingly not T cells. Homing of WT and CD53-/- purified B and T cells demonstrated that CD53-/- lymphocytes, particularly B cells, were incapable of efficiently homing to PLN. Hence, CD53 may be required for both expression of and signalling through L-selectin. We provide preliminary evidence that CD53 regulates L-selectin expression through a metalloproteinase, possibly a member of the ADAM family of proteases. Shedding of L-selectin from the surface of CD53-/- T cells was greatly accelerated as compared to WT, and could be largely inhibited by treatment with a broad matrix metalloproteinase inhibitor. We also showed impaired responses in a variety of inflammatory models including leukocyte/endothelial interactions, peritonitis and EAE in CD53-/- mice. This work shows that tetraspanins are essential not only for the regulation of integrins but also selectins.