Migrasomes are extracellular vesicles that form on the retraction fibers ofmigrating cells. In this study, we report the formation of migrasome-like vesi-cles enriched in tetraspanin 4 and containing cytoplasmic components inresponse to hypoosmotic stress. When migrating cells were subjected tohypoosmotic stress, vesicles with a size distribution of 0.5 to 2 lm formed onthe retraction fibers, and vanished in a few minutes. The vesicles are rich incholesterol, and their number was reduced when cells were pretreated withlipoprotein-deficient serum. The formation of migrasome-like vesicles uponhypoosmotic stress may provide biophysical cues regarding the cellularresponse to this external stimulus in cells and tissues.