[Background] Calcimycin is a structural unique pyrrole polyether antibiotic produced by Streptomyces chartreusis NRRL3882, and exhibits multiple biological effects, but the regulatory mechanism of calcimycin biosynthesis remains unclear. [Objective] To study on the function of the potential regulatory gene calR2 encoding LuxR family homologous protein in calcimycin biosynthesis gene cluster. [Methods] The calR2 gene on calcimycin biosynthesis gene cluster was disrupted by PCR-targeting, and metabolites of the ΔcalR2 mutant and complementation strain were analyzed by HPLC. The calcimycin biosynthesis gene expression levels of ΔcalR2 mutant compared with the wild-type strain was analyzed by RT-qPCR. [Results] HPLC analysis of the fermentation products showed that the ΔcalR2 mutant could not produce calcimycin and the complementation strain restored the production of calcimycin. Gene expression analysis showed that the transcription levels of some essential genes in calcimycin biosynthesis obviously declined in ΔcalR2 mutant. [Conclusion] The putative LuxR family homologous protein CalR2 acts as a positive regulator involved in calcimycin biosynthesis.