Sialic acids on vertebrate cell surfaces mediate many biological roles. Altered expression of certain sialic acid types or their linkages can have prognostic significance in human cancer. A classic but unexplained example is enhanced A2-6-sialylation on N-glycans resulting from overexpression of the Golgi enzyme B-galactoside:A2-6-sialyltransferase (ST6Gal-I). Previous data supporting a role for the resulting SiaA2-3GalB1-4GlcNAc (Sia6LacNAc) structure in tumor biology were based on in vitro studies in transfected carcinoma cells, in which increased Sia6LacNAc on B1-integrins enhanced their binding to ligands, and stimulated cell motility. Here, we examine for the first time the in vivo role of the ST6Gal-I enzyme in the growth and differentiation of spontaneous mammary cancers in mice transgenic for a mouse mammary tumor virus promoter-driven polyomavirus middle T antigen, a tumor in which B1-integrin function is important for tumorigenesis and in maintaining the proliferative state of tumor cells. Tumors induced in St6gal1-null animals were more differentiated compared with those in the wild-type background, both by histologic analysis and by protein expression profiles. Furthermore, we show the St6gal1-null tumors have selectively altered expression of genes associated with focal adhesion signaling and have decreased phosphorylation of focal adhesion kinase, a downstream target of B1-integrins. This first in vivo evidence for a role of ST6Gal-I in tumor progression was confirmed using a novel approach, which conditionally restored St6gal1 in cell lines derived from the null tumors. These findings indicate a role for ST6Gal-I as a mediator of tumor progression, with its expression causing a less differentiated phenotype, via enhanced B1-integrin function. [Cancer Res 2008;68(2):388-94]