The Gram-negative photoheterotrophic bacterium Dinoroseobacter shibae is a member of the high abundant marine Roseobacter group. In the ocean, OMVs have about the same abundance as bacteria, a distinct depth distribution, and contain DNA from a variety of marine bacterial taxa. In the present study, we determined the abundance, size, and ultrastructure of membrane vesicles of D. shibae and analysed the protein inventory of the soluble and membrane fractions of cells and vesicles in order to study the origin of OMV membranes and content. The proteomic analyses were complemented by fatty acid analyses.