The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP-ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs.