<jats:title>Abstract</jats:title> <jats:p>Combinatorial approach of adsorbent resin HP20 addition and metabolic profiling analysis were carried out to enhance ascomycin production. Under the optimal condition of 5 % m/v HP20 added at 24 h, ascomycin production was increased to 380 from 300 mg/L. To further rationally guide the improvement of ascomycin production, metabolic profiling analysis was employed to investigate the intracellular metabolite changes of Streptomyces hygroscopicus var. ascomyceticus FS35 in response to HP20 addition. A correlation between the metabolic profiles and ascomycin accumulation was revealed by partial least-squares to latent structures discriminant analysis, and 11 key metabolites that most contributed to metabolism differences and ascomycin biosynthesis were identified. Based on the analysis of metabolite changes together with their pathways, the potential key factors associated with ascomycin overproduction were determined. Finally, rationally designed fermentation strategies based on HP20 addition were performed as follows: 2 % v/v n-hexadecane was added at 24 h; 1.0 g/L valine was supplemented at 48 h; 1.0 g/L lysine was added at 72 h. The ascomycin production was ultimately improved to 460 mg/L, a 53.3 % enhancement compared with that obtained in initial condition. These results demonstrated that the combination of HP20 addition and metabolic profiling analysis could be successfully applied to the rational guidance of production improvement of ascomycin, as well as other clinically important compounds.