[Background] Hygrocins, a kind of naphthalene ansa antibiotics, are potential leading compounds for the chemical biosynthesis of novel drugs. However, under the common medium and fermentation conditions, the content of Hygrocin A in the cell is generally very low, and it is difficult to detect it directly. [Objective] In order to improve the yield of Hygrocin A from Streptomyces hygroscopicus ATCC 29253. [Methods] Effects of carbon source, nitrogen source, phosphate concentration, MgCl2 concentration, NaCl concentration and seed age on Hygrocin A production from S. hygroscopicus ATCC 29253 were studied by single factor and orthogonal test design. [Results] The optimal fermentation conditions were as follows: glucose 4.0 g/L, soybean cake meal 8.0 g/L, malt extract 10.0 g/L, K2HPO4 1.5 g/L, KH2PO4 1.5 g/L, NaCl 1.5 g/L, MgCl2 1.0 g/L; seed age 48 h; culture parameters: shaking speed 200 r/min, pH 6.8?7.0, bottled in 50 mL/250 mL, inoculated quantity 5%, incubated at 30 °C for 10 days. Under optimized conditions, the yield of Hygrocin A increased 500%, compared with its original medium M10, whereas at the same time the yield of Rapamycin decreased 95%. [Conclusion] The yield of Hygrocin A from S. hygroscopicus ATCC 29253 was significantly improved by optimizing the fermentation medium, which lay the foundation for studying the synthesis and application of Hygrocin A. At the same time, the yield of Rapamycin decreased significantly. This suggests that the metabolic flux of the two antibiotics can be adjusted intentionally by selecting the culture conditions, and then the metabolic regulation research for simultaneous expression of multiple antibiotics can be carried out.