Updated project metadata. The biogenesis and functions of Leptospira's extracellular vesicles (EVs) remain unclear. The study aimed to characterize EVs that were naturally released from intact leptospires during in vitro culture, as well as under conditions mimicking the host environment during infection, including temperature or osmolarity shift. The EVs were found to contain a group of proteins from the outer membrane and various cytoplasmic proteins, suggesting that they originated from the outer membrane and cytoplasm. These proteins were primarily involved in Leptospira's growth, survival, adaptation, and pathogenicity. Interestingly, the study revealed that Leptospira secreted multifunctional proteins through EVs but might maintain several virulence and virulence-associated proteins in their cells in response to temperature and osmotic stresses. Thus, native leptospiral EVs might act as decoys for the host immune system, while the virulence factors crucial for direct interaction with host components might be preserved in whole cells. This knowledge may contribute to understanding the role of EVs in the pathogenicity of Leptospira and the development of vaccines against leptospirosis in the future.