Abstract The immune scavenger protein DC‐SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC‐SIGN have not been reported. Herein, we report the development and application of a mass‐spectrometry‐based approach to both uncover and characterise the effects of O‐glycans on the stability of DC‐SIGN. We first quantify the Core 1 and 2 O‐glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O‐glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas‐phase stability of the DC‐SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC‐SIGN is better correlated with the number of glycosylation sites.