Inflammatory Bowel Disease (IBD) is a chronic disease characterised by episodic and disabling inflammation of the gut.  The gut microbiota is central to the pathogenesis of IBD and consistent with this the onset of active disease is associated with an innate immune response that includes the sequestration of iron to suppress microbial growth.  However, with the notable exception of the Proteobacteria, little is known about how this “nutritional immunity” affects the growth of fastidious gut bacteria and their ability to persist in the gut.  The human gut bacterium Bacteroides vulgatus is widely considered to be a pathobiont – a symbiont that can cause pathology in response to host and/or environmental triggers – and it has been repeatedly implicated in the pathogenesis of IBD.  Using a custom high-throughput anaerobic culturing platform we determined B. vulgatus has an absolute nutritional requirement for iron and can utilise a wide range of host derived iron sources including haemin, haemoglobin, transferrin, lactoferrin and ferritin to support growth under low iron conditions.  B. vulgatus is genetically recalcitrant and we thus developed an efficient mutagenesis strategy that exploits the promiscuous RP4 conjugative transfer system and the mariner transposon.  We recovered >39,000 mutants and achieved a theoretical coverage of >99% of the genome.  Following penicillin-based enrichment to select for B. vulgatus mutants defective in haemin utilization we identified no less than three distinct genetic loci that underpin haem iron utilisation.  Two of these loci encoded the TonB dependent outer membrane receptor and TonB-ExbBD energy transduction system respectively, while the third had not previously been implicated in haem iron utilisation.  We analysed previously published INSeq mutagenesis data for Bacteroides thetaiotaomicron and Bacteroides ovatus and determined these loci are necessary for optimal fitness during in vitro and in vivo growth.  Our study has provided the first mechanistic insight into iron acquisition by B. vulgatus.  We contend that genetic dissections offer an approach complementary to genomics to elucidate the functional capacity of fastidious gut bacteria.