Abstract:[Objective] We revealed the function of the ArsR (Arsenical resistance regulator) family regulator XanR3 in xantholipin biosynthetic gene cluster. [Methods] Genetically, the xanR3 disrupted mutant CGF03 and complementary strain CGF06 were constructed by conjugation between S. flavogriseus SIIA-A02191 and Escherichia coli ET12567/pUZ8002. The operons within xantholipin biosynthesis cluster were grouped by RT-PCR and the transcriptional level was analyzed in the mutant CGF-3 by quantitative real-time RT-PCR. [Results] Inactivation of the xanR3 gene dramatically reduced xantholipin biosynthesis, and its complementation partially restored xantholipin production. Totally, genes in xantholipin biosynthetic gene cluster were assigned into 18 co-transcription units, and the disruption of xanR3 directly down-regulated the transcription of four operons as xanO1-xanS1, xanB1-xanB3, xanT-xanO9 and xanO10-xanC3.[Conclusion] XanR3, a ArsR family transcriptional regulator, is an activator of xantholipin biosynthesis.