The novel exopolysaccharide Ebosin produced by Streptomyces sp. 139 has remarkable in vivo antirheumatic arthritis activity, and its biosynthesis gene cluster (ste) consisting of 27 ORFs has been identified. The present investigation focused on the function of ste1 gene. Database searching revealed that Ste1 is homologous to the GntR family regulator originated from microbes. To confirm its function in Ebosin biosynthesis, the gene was disrupted. The mutant strain Streptomyces sp. 139 D1 was found to have a much higher Ebosin production than that of the wild-type strain, whilst the complementation strain Streptomyces sp. 139 C1 showed a decrease in the exopolysaccharide produced. Real-time qPCR analysis indicated that in the mutant strain Streptomyces sp. 139 D1, transcription levels of gene ste5, ste8, and ste17 increased significantly compared with those in the wild-type strain. The electrophoretic mobility shift assay demonstrated that Ste1 binds with higher affinity to the promoter 1 and 3 regions in the ste gene cluster. It is concluded that ste1 plays the negative regulation as a transcription repressor during Ebosin biosynthesis. Growing on minimal agar medium supplemented with glucose and R2YE agar medium, the mutant strain Streptomyces sp. 139 D1 exhibited a Bld phenotype.