Sulfate-reducing bacteria are known to mediate dolomite formation under hypersaline conditions, but details of the crystal nucleation process are still poorly constrained. Our laboratory study demonstrates for the fi rst time that Desulfobulbus mediterraneus, a marine sulfate-reducing bacterium, mediates primary precipitation of Mg-rich dolomite under anoxic conditions in media replicating modern seawater chemistry at low temperature (21 °C). Precipitation of crystals was associated with extracellular polymeric substances in a monospecifi c biofi lm, providing templates for nucleation by altering the molar Mg/Ca ratio. After initial nucleation of single nanospherulites (~50 nm), growth was mediated by aggregation, resulting in spherulites of ~2-3 μm in diameter. Nucleation led to differences in Mg/Ca ratios and δ44/40Ca values among the organic material (i.e., biofi lm including cells and extracellular polymeric substances; 0.87 ± 0.01 [2 SD] and 0.48‰ ± 0.11‰ [2 SE], respectively), the crystals (1.02 ± 0.11 [2 SD] and <-0.08‰ ± 0.24‰ [2 SE], respectively), and the liquid bulk medium after mineral precipitation (4.53 ± 0.04 [2 SD] and 1.10‰ ± 0.24‰ [2 SE], respectively). These data indicate a two-step fractionation process involved in the sequestration of Ca from the solution into the crystal lattice of the mineral precipitated. Our results demonstrate the capability of extracellular polymeric substances to overcome kinetic inhibition, fostering the forma tion of kinetically less favorable Mg-rich dolomite, and they also question the applicability of the Ca isotopic system as a proxy for paleogeochemistry of seawater.