Natamycin is a polyene macrolide antibiotic and widely used as a natural food preservative. Fungal elicitor had positive effects on the natamycin biosynthesis in Streptomyces natalensis HW-2. However, the global gene expression in response to fungal elicitor is not still reported. In the study, RNA-Seq was used to check the change of transcriptome by fungal elicitor in S. natalensis HW-2. The results showed that there were 1265 differential expression genes (DEGs) at 40 h and 2196 DEGs at 80 h. Most of the genes involved in natamycin biosynthesis were upregulated. KEGG pathway analysis showed that fungal elicitor had strong effects on the transcriptional levels of genes related to branch-chained amino acid (BCAA) metabolism. There were 23 upregulated or downregulated DEGs involved in BCAA biosynthesis and degradation at 40 h and 80 h. To confirm whether the improvement of BCAA biosynthesis could produce more natamycin, metabolic engineering was used to homologously overexpress the gene ilvH which encoded the regulatory subunit of acetolactate synthase (ALS) in S. natalensis. The results showed that overexpression of ilvH in S. natalensis HW-2 increased natamycin production to 1.25 g/L in the flask, which was a 32% increase compared with that of the parent strain. Real-time quantitative PCR analysis showed that the transcriptional level of ilvH in mutant strain S. natalensis ZS101 was significantly increased. Acetyl-CoA content was also raised. The results suggested that the fungal elicitor enhanced natamycin biosynthesis by improving precursor supply via BCAA metabolism. This study will open a new avenue for enhancing natamycin production by metabolic engineering and adding fungal elicitor.