Iron-dependent regulator (IdeR), a metal ion-activated pleiotropic transcription factor, plays a critical role in maintaining the intracellular iron homeostasis in Mycobacteria, which is important for the normal growth of the cells. This study was initially performed in an attempt to elucidate all potential interactions between the various domains of IdeR that occur in living mycobacterial cells. This led to a hitherto unidentified self-association for the SH3-like domain of IdeR. Further studies demonstrate that the SH3-like domain interacts with different partners in the dimeric forms of IdeR depending on the levels of metal ions in the environment: it undergoes inter-subunit self-association in the metal-free DNA-non-binding form, but interacts with the N-terminal domain in the metal-bound DNA-binding form in an intra-subunit manner to finely modulate the transcription repression activity of IdeR. Our more detailed mapping studies reveal that the SH3-like domain uses an overlapping surface to participate in these two interactions, which therefore occur in a mutually exclusive fashion. This novel mechanism would allow an effective and cooperative interconversion between the two functional forms of IdeR. Our data also demonstrate that a disturbance of the interactions involving the SH3-like domain impairs the transcription repression activity of IdeR and delays the growth of mycobacterial cells.