Within the host, free iron is limited and its acquisition by infecting mycobacteria is thought to play a major role in the development of pathogenesis. Moreover, iron is an essential element for most organisms and functions as a prosthetic group in key metabolic pathways such as electron transport, oxidative stress and transmembrane transport. Mycobacteria have developed effective acquisition systems that involve the production of the iron-chelating molecules (siderophores) mycobactin, exochelin and carboxymycobactin.The aim was to develop iron deficient mycobacterial cultures and utilise both genomic and proteomic approaches to study the role of iron in mycobacterial cell metabolism. Conditions for iron deficient cultures were optimised for the fast growing M. smegmatis and applied to slow growing M. avium ssp. avium and M. avium ssp. paratuberculosis. Iron deficiency in these cultures was confirmed by Reverse Transcription Quantitative Real Time PCR and an exochelin bioassay. Proteomic profiles of iron deficient cultures have been generated using two-dimensional electrophoresis and compared with those grown under normal culture conditions. Differentially expressed proteins were analysed using mass spectrometry and N-terminal protein sequencing. Their role in cell metabolism and their potential role in pathogenicity will be discussed.