The oxygen regulator Fnr is part of the regulatory cascade in Bacillus subtilis for the adaptation to anaerobic growth conditions. In vivo complementation experiments revealed the essential role of only three cysteine residues (C227, C230, C235) at the C-terminus of B. subtilis Fnr for the transcriptional activation of the nitrate reductase operon (narGHJI) and nitrite extrusion protein gene (narK) promoters. UV/VIS, electron paramagnetic spin resonance (EPR) and Mössbauer spectroscopy experiments in combination with iron and sulphide content determinations using anaerobically purified recombinant B. subtilis Fnr identified the role of these three cysteine residues in the formation of one [4Fe-4S]2+ cluster per Fnr molecule. The obtained Mössbauer parameters are supportive for a [4Fe-4S]2+ cluster with three cysteine ligated iron sites and one non-cysteine ligated iron site. Gel filtration experiments revealed a stable dimeric structure for B. subtilis Fnr which is independent of the presence of the [4Fe-4S]2+ cluster. Gel mobility shift and in vitro transcription assays demonstrated the essential role of an intact [4Fe-4S]2+ cluster for promoter binding and transcriptional activation. An amino acid exchange introduced in the proposed αD-helix of B. subtilis Fnr (G149S) abolished its in vivo and in vitro activities indicating its importance for intramolecular signal transduction. The clear differences in the localization and coordination of the [4Fe-4S] cluster and in the organization of the oligomeric state between Escherichia coli and B. subtilis Fnr indicate differences in their mode of action.