Cell adhesion molecules regulate a variety of endothelial cell functions such as migration, response to inflammation, and angiogenesis. Recently, activated leukocyte cell adhesion molecule (ALCAM), a member of the Ig superfamily, has been detected in the primitive subsets of hematopoietic cells and endothelial cells during embryogenesis. ALCAM supports the development of hematopoietic cells as well as enhancing capillary tube formation in vitro. Here, we isolated a novel soluble isoform of ALCAM (sALCAM) that is produced via alternative splicing. sALCAM contains the single amino-terminal Ig-like domain of ALCAM and lacks a transmembrane domain. When expressed in cultured cells, sALCAM was properly secreted into the media. Both ALCAM and sALCAM are expressed in a variety of cultured human endothelial cells. Notably, their transcripts were differentially regulated in human microvascular endothelial cells (HMVEC) upon tumor necrosis factor-alpha stimulation. ALCAM significantly enhanced tube formation of endothelial-like yolk sac cells on Matrigel, whereas it inhibited their migration in vitro. sALCAM completely abolished these effects of ALCAM. Furthermore, sALCAM enhanced migration of mock-transfected endothelial-like yolk sac cells that do not express ALCAM, indicating that sALCAM has an independent effect on cell migration in addition to modulating ALCAM function. In addition, sALCAM significantly enhanced migration of HMVEC, whereas it inhibited tube formation of HMVEC on Matrigel. sALCAM demonstrated an ability to bind ALCAM and partially inhibited ALCAM-ALCAM homophilic interactions. Taken together, these data characterize a novel soluble isoform of ALCAM that may have ALCAM-dependent and ALCAM-independent functions, providing further insights regarding the role of this adhesion molecule in the regulation of endothelial cell function.