Abstract This study elucidates ST6Gal-I, a sialyltransferase, as a functional driver of a cancer stem cell (CSC) phenotype regulated by the stem cell transcription factor Sox2. ST6Gal-I is upregulated in 98% of ovarian cancers (OC); it functions to add an α2-6 sialic acid, a large, negatively charged sugar, to N-glycosylated proteins bound for the cell surface. Normal differentiated epithelia have very low expression of ST6Gal-I, however, expression is turned on in stem cell compartments and transformed tissues. Furthermore, our work has shown that ST6Gal-I plays a causal role in conferring hallmark CSC properties including greater tumor-initiating capabilities, promotion of recurrence, and resistance to tumor-associated stressors like chemotherapies, serum deprivation, and hypoxia. Even given this stark upregulation and apparent functional import of ST6Gal-I in cancers, very little work has been done to identify the transcriptional driver of ST6Gal-I expression. We identified that Sox2 and ST6Gal-I are both part of one of the most commonly enriched amplicons in human cancer, amplicon 3q26. We then examined the TCGA database and found that these two genes are co-amplified in 48/73 cancer cohorts, including ovarian cancer. In addition to being genetically co-amplified, Sox2 has been shown to bind to the promoter of ST6Gal-I, though no further analyses have been performed to assess a functional link. Herein we address a novel, glycosylation-dependent mechanism that drives a CSC phenotype. We hypothesize that Sox2, a key stem-associated transcription factor in CSCs, directly induces expression of ST6Gal-I. In our ovarian cancer cell model systems, we forced overexpression or knockdown of Sox2 and found that Sox2 expression directly correlated with ST6Gal-I expression. Overexpression or knockdown of Sox2 resulted in up- or down- regulation of CSC markers Oct4 and Nanog respectively. We then forced ST6Gal-I knockdown to inhibit the Sox2-induced ST6Gal-I upregulation and found that this prevented the enhanced expression of Oct4 and Nanog. Here we show that Sox2 requires ST6Gal-I expression to promote a CSC phenotype. Citation Format: Kaitlyn A. Dorsett, Susan L. Bellis. Sox2 drives ST6Gal-I expression and activity to promote a CSC phenotype in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1129.