CD99-like 2 (CD99L2 [L2]) is a highly glycosylated 52-kDa type 1 membrane protein that is important for leukocyte transendothelial migration (TEM) in mice. Inhibiting L2 using function-blocking Ab significantly reduces the recruitment of leukocytes to sites of inflammation in vivo. Similarly, L2 knockout mice have an inherent defect in leukocyte transmigration into sites of inflammation. However, the role of L2 in inflammation has only been studied in mice. Furthermore, the mechanism by which it regulates TEM is not known. To study the relevance to human inflammation, we studied the role of L2 on primary human cells in vitro. Our data show that like PECAM and CD99, human L2 is constitutively expressed at the borders of endothelial cells and on the surface of leukocytes. Inhibiting L2 using Ab blockade or genetic knockdown significantly reduces transmigration of human neutrophils and monocytes across endothelial cells. Furthermore, our data also show that L2 regulates a specific, sequential step of TEM between PECAM and CD99, rather than operating in parallel or redundantly with these molecules. Similar to PECAM and CD99, L2 promotes transmigration by recruiting the lateral border recycling compartment to sites of TEM, specifically downstream of PECAM initiation. Collectively, our data identify a novel functional role for human L2 in TEM and elucidate a mechanism that is distinct from PECAM and CD99.