CD99 is known to be involved in the regulation of cell-cell adhesion. However, it remains unclear whether CD99 controls cell-extracellular matrix adhesion. In this study, the effects of CD99 activation on cell-extracellular matrix adhesion were investigated. It was found that engagement of CD99 with the stimulating antibody YG32 downregulated the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV in a dose-dependent manner. The CD99 effect on cell-ECM adhesion was inhibited by overexpression of the dominant negative form of CD99 or CD99 siRNA transfection. Treatment of cells with $Mn^{2+}$ or by ${eta}_1$ integrin-stimulating antibody restored the inhibitory effect of CD99 on cell-ECM adhesion. Cross-linking CD99 inactivated ${eta}_1$ integrin through conformational change. CD99 activation caused dephosphorylation at Tyr-397 in FAK, which was restored by the ${eta}_1$ stimulating antibody. Taken together, these results provide the first evidence that CD99 inhibits cell-extracellular matrix adhesion by suppressing ${eta}_1$ integrin affinity.