Vitamin B12(cobalamin, herein B12) is an essential cofactor involved in amino acid synthesis and carbon resupply to the TCA cycle for most prokaryotes, eukaryotic microorganisms, and animals. Despite being required by most, B12is produced by only a minor fraction of prokaryotes and therefore leads to complex interaction between prototrophs and auxotrophs. However, it is unknown how B12is provided by prototrophs to auxotrophs. In this study, 33 B12prototrophic alphaproteobacterial strains were grown in co-culture withThalassiosira pseudonana, a B12auxotrophic diatom, to determine the bacterial ability to support the growth of the diatom by sharing B12. Among these strains, 18 were identified to share B12with the diatom, while nine were identified to retain B12and not support growth of the diatom. The other bacteria either shared B12with the diatom only with the addition of substrate or inhibited the growth of the diatom. Extracellular B12measurements of B12-provider and B12-retainer strains confirmed that the cofactor could only be detected in the environment of the tested B12-provider strains. Intracellular B12was measured by LC-MS and showed that the concentrations of the different B12-provider as well as B12-retainer strains differed substantially. Although B12is essential for the vast majority of microorganisms, mechanisms that export this essential cofactor are still unknown. Our results suggest that a large proportion of bacteria that can synthesise B12de novocannot share the cofactor with their environment.