Abstract Background Small extracellular vesicles (sEV) are closely associated with the development and metastasis of many types of mammalian cancer. Glycoconjugates are highly expressed on sEV and play important roles in sEV biogenesis and their interaction with other cells. However, the study on vesicular glycoconjugates are far behind proteins and nucleic acids. Especially, the functions of sialic acids which are the terminal components of glycoconjugates, are poorly understood in sEV. Methods Sialic acid levels on sEV from plasma and bladder cancer cells were determined by ELISA and lectin blotting. Effects of sialylation on sEV uptake were determined by flow cytometry. Vesicular glycoproteins bearing sialic acids responsible for sEV uptake was identified by proteomics and density gradient centrifugation, and their site-specific sialylation functions were assayed by N-glycosylation site mutation. Effects of integrin β1 bearing sialic acids on the pro-metastatic function of sEV in vivo were explored using Balb/c nu/nu mice. Results (1) Increased sialic acid levels were observed in sEV from malignant bladder cancer cells. (2) Elimination of sialic acids on sEV impaired sEV uptake by recipient cells. (3) Vesicular integrin β1 bearing sialic acids was identified to play a key role in sEV uptake. (4) Desialylation of the hybrid domain of vesicular integrin β1 inhibited its binding to matrix fibronectin, and reduced sEV entry into recipient cells. (5) Sialylation on integrin β1 affected pro-metastatic function of sEV in Balb/c nu/nu mice. Conclusions Taken together, our findings indicate important functional roles of sialic acids in sEV uptake and reprogramming plasticity of surrounding normal epithelial cells. Graphical Abstract