P-selectin glycoprotein ligand-1 (PSGL-1) is an adhesive molecule that is known to be a ligand for P-selectin. An anti-adhesive property of PSGL-1 has not been previously reported. In this study, we show that PSGL-1 expression is anti-adhesive for adherent cells and we have elucidated the underlying mechanism. Overexpression of PSGL-1 induced cell rounding and floating in HEK293T cells. Similar phenomena were demonstrated in other adherent cell lines with overexpression of PSGL-1. PSGL-1 overexpression inhibits access of antibodies to cell surface molecules such as integrins, HLA and CD25. Cells transfected with PSGL-1 deletion mutants that lack a large part of the extracellular domain and chimeric construct expressing extracellular CD86 and intracellular PSGL-1 only showed rounded morphology, but there are no floating cells. These results indicated that PSGL-1 causes steric hindrance due to the extended structure of its extracellular domain that is highly O-glycosylated, but intracellular domain also has some effect on cell rounding. This study implies that PSGL-1 has Janus-faced functions, being both adhesive and anti-adhesive. J. Cell. Biochem. 114: 1271–1285, 2013. © 2013 Wiley Periodicals, Inc.