The biosynthesis of antibiotics is controlled by cascade regulation involving cluster-situated regulators (CSRs) and pleiotropic regulators. Three CSRs have been identified in the jadomycin biosynthetic gene cluster, including one OmpR-type activator (JadR1) and two TetR-like repressors (JadR and JadR2). To examine their interactions in jadomycin biosynthesis, a series of mutants were generated and tested for jadomycin production. We noticed that jadomycin production in the jadR-jadR2 double mutant was increased dramatically compared with either single mutant. Transcriptional analysis showed that jadR and jadR2 act synergistically to repress jadomycin production by inhibiting the transcription of jadR1. Furthermore, jadR and jadR2 reciprocally inhibit each other. The complex interactions among these three CSRs may provide clues for the activation of the jadomycin gene cluster, which would otherwise remain silent without stimulation from stress signals.