α-Secretase processing of APP has recently gained more interest, highlighting its potential as a therapeutic target to prevent Alzheimer’s disease (AD). We have previously shown that O-GlcNAcylation stimulates α-secretase processing of APP, concomitantly with decreased Aβ secretion. O-GlcNAcylation has previously been linked to AD since the levels of O-GlcNAcylated proteins are decreased in AD brains. Here, we have further investigated the mechanism behind α-secretase processing of APP in response to increased O-GlcNAcylation. Our results shown that APP is not O-GlcNAcylated during the conditions used in this study. Instead, we demonstrate that the α-secretase ADAM10 is O-GlcNAcylated and that APP cell surface localization is enhanced in response to increased O-GlcNAcylation. Furthermore, the effects of O-GlcNAcylation on APP processing are cell-type specific, only affecting sAPPα secretion in neuroblastoma cell-lines.