Precursor engineering is an effective strategy for the overproduction of secondary metabolites. The polyene macrolide rimocidin, which is produced by Streptomyces rimosus M527, exhibits a potent activity against a broad range of phytopathogenic fungi. It has been predicted that malonyl-CoA is used as extender units for rimocidin biosynthesis. Based on a systematic analysis of three sets of time-series transcriptome microarray data of S . rimosus M527 fermented in different conditions, the differentially expressed acc sr gene that encodes acetyl-CoA carboxylase (ACC) was found. To understand how the formation of rimocidin is being influenced by the expression of the acc sr gene and by the concentration of malonyl-CoA, the acc sr gene was cloned and over-expressed in the wild-type strain S . rimosus M527 in this study. The recombinant strain S . rimosus M527-ACC harboring the over-expressed acc sr gene exhibited better performances based on the enzymatic activity of ACC, intracellular malonyl-CoA concentrations, and rimocidin production compared to S . rimosus M527 throughout the fermentation process. The enzymatic activity of ACC and intracellular concentration of malonyl-CoA of S . rimosus M527-ACC were 1.0- and 1.5-fold higher than those of S . rimosus M527, respectively. Finally, the yield of rimocidin produced by S . rimosus M527-ACC reached 320.7 mg/L, which was 34.0% higher than that of S . rimosus M527. These results confirmed that malonyl-CoA is an important precursor for rimocidin biosynthesis and suggested that an adequate supply of malonyl-CoA caused by acc sr gene over-expression led to the improvement in rimocidin production.