Differentiation of monocytes into macrophages is accompanied by increased cell adhesiveness, due in part to the activation of α4β1 integrins. Here we report that the sustained α4β1 activation associated with macrophage differentiation results from expression of β1 integrin subunits that lack α2-6-linked sialic acids, a carbohydrate modification added by the ST6Gal-I sialyltransferase. During differentiation of U937 monocytic cells and primary human CD14+ monocytes, ST6Gal-I is down-regulated, leading to β1 hyposialylation and enhanced α4β1-dependent VCAM-1 binding. Importantly, ST6Gal-I down-regulation results from cleavage by the BACE1 secretase, which we show is dramatically up-regulated during macrophage differentiation. BACE1 up-regulation, ST6Gal-I shedding, β1 hyposialylation, and α4β1-dependent VCAM-1 binding are all temporally correlated and share the same signaling mechanism (protein kinase C/Ras/ERK). Preventing ST6Gal-I down-regulation (and therefore integrin hyposialylation), through BACE1 inhibition or ST6Gal-I constitutive overexpression, eliminates VCAM-1 binding. Similarly, preventing integrin hyposialylation inhibits a differentiation-induced increase in the expression of an activation-dependent conformational epitope on the β1 subunit. Collectively, these results describe a novel mechanism for α4β1 regulation and further suggest an unanticipated role for BACE1 in macrophage function. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.