Iron-ligand complexes in marine waters are structurally diverse, and much is yet to be learned of their structure. Heme, a form of porphyrin bound iron, has been detected at nanomolar concentrations in seawater, but the bioavailability and utilization of heme by marine organisms and its biogeochemical significance has rarely been examined. Here we investigate the molecular physiological basis of marine bacterial heme uptake and assimilation pathways that have been inferred bioinformatically. We show that putatively annotated heme uptake gene clusters in isolates of the marine Roseobacter clade are in fact viable systems for acquiring iron from heme and porphyrin-like ligands. A particular isolate, Silicibacter sp. TrichCH4B, can maintain growth solely on ferric-porphyrin complexes, and its putative heme uptake and utilization system is up-regulated under iron stress. We also describe our efforts in generating Roseobacter genetic mutants with inactivated heme uptake and utilization genes. This functional characterization is a step forward in understanding the specific biogeochemical origin, role, and fate of heme-based molecules in seawater.