Sporulation of Bacilli is a developmental process that provides long-term viability in unfavorable environments. Recently, biogenesis of extracellular vesicles (EVs) from Bacilli has also been reported to participate in various physiological and pathogenic phenomena. In this study, EVs were isolated from vegetative and sporulating Bacillus subtilis cells and characterized using mass spectrometry (MS)-based proteomics, microscopy, and fluorescence spectrophotometry. The microscopic approach demonstrated that both vegetative and sporulating cells produce EVs. In the proteomic analysis, 156 proteins were identified with statistical significance in EVs collected at the vegetative phase and 185 proteins in EVs shed during sporulation. The two EV cargos showed qualitatively and quantitatively different proteome patterns. Sporulation-associated proteins had greater abundances in EVs at the sporulation stage. Additionally, a fusion-like event of EVs with B. subtilis cells was observed by a fluorescence de-quenching assay. Based on these observations, B. subtilis EVs are proposed to support intercellular communication and sporulation.