AbstractSoybean oil is an important carbon source in kitasamycin fermentation by Streptomyces kitasatoensis. In this study, three different surfactants, Tween 80, Tween 85, and sodium dodecyl sulfate (SDS), were added in the fermentation medium to improve soybean oil utilization. Results indicated that all of these surfactants promote kitasamycin biosynthesis. When 0.5 g/L SDS was added at the beginning of fermentation, kitasamycin production increased by 55% and A5 content improved by 12%, compared with the control treatment (i.e., no surfactant added). Oil consumption rate and lipase activity were also improved in the presence of SDS, producing more organic acids benefiting kitasamycin biosynthesis. High butyric acid concentration in the fermentation medium containing SDS repressed C‐3 acetylation and promoted A5 component accumulation. Additionally, utilization of oil components by S. kitasatoensis was altered. Specifically, linoleic acid was primarily used in the fermentation process with SDS, whereas oleic acid was primarily used in the fermentation process where no surfactant had been added.