Salinomycin, a polyether antibiotic produced by Streptomyces albus, is widely used in animal husbandry as an anticoccidial drug and growth promoter. Situated within the salinomycin biosynthetic gene cluster, slnR encodes a LAL-family transcriptional regulator. The role of slnR in salinomycin production in S. albus was investigated by gene deletion, complementation, and overexpression. Gene replacement of slnR from S. albus chromosome results in almost loss of salinomycin production. Complementation of slnR restored salinomycin production, suggesting that SlnR is a positive regulator of salinomycin biosynthesis. Overexpression of slnR in S. albus led to about 25 % increase in salinomycin production compared to wild type. Quantitative RT-PCR analysis revealed that the expression of most sal structural genes was downregulated in the Delta slnR mutant but upregulated in the slnR overexpression strain. Electrophoretic mobility gel shift assays (EMSAs) also revealed that SlnR(DBD) binds directly to the three intergenic regions of slnQ-slnA1, slnF-slnT1, and slnC-slnB3. The SlnR binding sites within the three intergenic regions were determined by footprinting analysis and identified a consensus-directed repeat sequence 5'-ACCCCT-3'. These results indicated that SlnR modulated salinomycin biosynthesis as an enhancer via interaction with the promoters of slnA1, slnQ, slnF, slnT1, slnC, and slnB3 and activates the transcription of most of the genes belonging to the salinomycin gene cluster but not its own transcription.