Tetraspanins regulate key processes in immune cells; however, the function of the leukocyte-restricted tetraspanin CD53 is unknown. Here we show that CD53 is essential for lymphocyte recirculation. Lymph nodes of Cd53 mice were smaller than those of wild-type mice due to a marked reduction in B cells and a 50% decrease in T cells. This reduced cellularity reflected an inability of Cd53 B and T cells to efficiently home to lymph nodes, due to the near absence of L-selectin from Cd53 B cells and reduced stability of L-selectin on Cd53 T cells. Further analyses, including on human lymphocytes, showed that CD53 stabilizes L-selectin surface expression and may restrain L-selectin shedding via both ADAM17-dependent and ADAM17-independent mechanisms. The disruption in lymphocyte recirculation in Cd53 mice led to impaired immune responses dependent on antigen delivery to lymph nodes. Together these findings demonstrate an essential role for CD53 in lymphocyte trafficking and immunity.