In T lymphocytes, Ca2+ release-activated Ca2+ (CRAC) channels composed of Orai1 subunits trigger antigen-induced gene expression and cell proliferation through the NFAT pathway. We evaluated the requirement of CRAC channel function for lymphocyte homing using expression of a dominant-negative Orai1-E106A mutant to suppress Ca2+ signaling. To investigate homing and motility of human lymphocytes in immunocompromised mouse hosts, we transferred human lymphocytes either acutely or following stable engraftment after a second transfer from the same blood donor. Human and mouse lymphocyte homing was assessed and cells were tracked within lymph nodes by two-photon microscopy. Our results demonstrate that human T and B lymphocytes home into and migrate within the lymph nodes of immunocompromised NOD.SCID mice similar to murine lymphocytes. Human T and B cells colocalized in atrophied or reconstituted mouse lymph nodes, where T cells migrated in a random walk at velocities of 9-13 μm/min and B cells at 6 μm/min. Expression of Orai1-E106A inhibited CRAC channel function in human and mouse T cells and prevented homing from high endothelial venules into murine lymph nodes. Ca2+ signals induced by CCL21 were also inhibited in T cells expressing Orai1-E106A. With CRAC channels inhibited, the high-affinity form of LFA-1 failed to become active, and T cells failed to migrate across endothelial cells in a transwell model. These results establish a requirement for CRAC channel-mediated Ca2+ influx for T cell homing to lymph nodes mediated by high-affinity integrin activation and chemokine-induced transendothelial migration.