Alterations of the glycosylation machinery are a common event in cancer that leads to the synthesis of aberrant glycan structures by tumor cells. Extracellular vesicles (EVs) play a modulator role in cancer communication and progression, and interestingly, several tumor-associated glycans have already been identified in cancer EVs. Nevertheless, the impact of 3D cellular architecture in the selective packaging of glycans into EVs has never been addressed. In this work, we have measured the capacity of gastric cancer cell lines carrying different glycosylation patterns in producing and releasing EVs when cultured under conventional 2D monolayer or in 3D culture conditions. Furthermore, we have identified the proteomic content and the presence of specific glycan patterns in the EVs produced by these cells, depending on their culture condition. We have observed that although the protein content of the analysed EVs was mostly conserved, an EV differential packaging of specific proteins and glycans was found, depending on the cell culture condition. Cellular component and pathway analysis have revealed different signatures on the EVs released by 2D and 3D cultured cells, suggesting distinct biological functions.