The polypeptide core of the integrin beta1 subunit (beta1) is glycosylated sequentially in the endoplasmic reticulum and the Golgi complex to form beta1 precursor and mature beta1, respectively. The beta1 precursor to mature beta1 conversion, termed beta1 maturation, regulates the cell surface levels and function of beta1-containing integrins, beta1 integrins. Here we demonstrate that the human alkaline ceramidase 2 (ACER2), a Golgi enzyme, regulates beta1 maturation by controlling the generation of sphingosine. ACER2 overexpression inhibited beta1 maturation, thus leading to a decrease in the levels of mature beta1 in T-REx HeLa cells, whereas RNA interference-mediated knockdown of ACER2 enhanced beta1 maturation in MCF-7 cells. ACER2 overexpression decreased the cell surface levels of beta1 integrins, thus inhibiting cell adhesion to fibronectin or collagen, whereas ACER2 knockdown has the opposite effects. Treatment with all-trans retinoic acid (ATRA) increased both the expression of ACER2 and the generation of sphingosine in HeLa cells and inhibited beta1 maturation. ACER2 knockdown attenuated the inhibitory effects of ATRA on both beta1 maturation and cell adhesion. In contrast, treatment with phorbol myristate acetate (PMA), a protein kinase C activator, decreased the expression of ACER2 and sphingosine in T-REx HeLa cells, thus enhancing beta1 maturation. ACER2 overexpression inhibited the stimulatory effects of PMA on both beta1 maturation and cell adhesion. These results suggest that the ACER2/sphingosine pathway plays an important role in regulating beta1 maturation and cell adhesion mediated by beta1 integrins.