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[Objective] Macrolactins (MLNs) are a class of 24-membered macrolides formed by cyclization of polyketones, which have a variety of biological activities such as antibacterial, antifungal, antiviral, and anti-tumor. However, due to the low yield of macrolides in traditional fermentation, its development and application are limited. Therefore, this article aims to increase the yield of MLNs synthesized by Bacillus siamensis K53, in order to provide scientific basis for the mass production of MLNs. [Method] Based on previous research, single factor experiments was used to study the effects of carbon, nitrogen and amino acids on the MLNs yield of strain K53 and to determine the key nutritional factors. Response surface analysis was used to optimize the key nutritional factors for obtaining the optimal parameters in fermenting MLNs. [Result] In the single factor experiment, glucose was the optimal carbon source in the fermentation medium of strain K53. The yeast extract and (NH4)2SO4 were the optimal organic and inorganic nitrogen sources, respectively. The addition of histidine and valine increased the yield of MLNs. In response surface optimization, the optimal fermentation medium for strain K53 was 22.0 g/L glucose, 3.0 g/L (NH4)2SO4, 1.0 g/L yeast extract, 6.1 g/L valine, and 5.9 g/L histidine. The valine and histidine were precursors for the synthesis of MLNs. The MLNs yield in this medium reached 5.37×10² μg/mL, which was very close to the maximum value (5.46×10² μg/mL) predicted by theory, and 4.10 times higher than that in the non-optimized medium (1.31×10² μg/mL). [Conclusion] The optimized medium formula by response surface methodology is conducive to the MLNs yield of the strain K53, and the results provide theoretical bases for the consequent development and application of the strain K53. [ABSTRACT FROM AUTHOR]