Streptomyces coelicolor is a model organism for the study of Streptomyces, a genus of Gram-positive bacteria that undergoes a complex life cycle and produces an enormous repertoire of bioactive metabolites and extracellular enzymes. This study investigated the production and characterization of membrane vesicles (MVs) in liquid cultures of S. coelicolor M145 from a structural and biochemical point of view using microscopic, physical and -omics analyzes. Two main populations of MVs, F3 and F4 MVs, with different size and cargo were isolated and purified. S. coelicolor MV cargo is complex and contains many “messages” such as proteins and metabolites. A total of 166 proteins involved in cell metabolism and differentiation, molecular processing and transport was identified in MVs. In particular, a subset of these proteins was protected from the degradation after proteinase K treatment, indicating their localization inside the vesicles. Vesicle proteome includes many stress response proteins which also play an important role in S. coelicolor morpho- physiological differentiation. Moreover, MVs packaged with an array of metabolites such as antibiotics, vitamins, amino acids and components of carbon metabolism. The analysis of S. coelicolor MV cargo will provide informations to elucidate their biogenesis and functions