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Cycloheptylprodigiosin exhibits potent anticancer activities through a unique mechanism involving the induction of severe Golgi stress, a previously unrecognized cell death pathway. In order to achieve higher fermentation yield of prodiginines from marine bacterium <i>Spartinivicinus ruber</i> MCCC 1K03745^T, the fermentation parameters including incubation time and original pH of culture medium were investigated. Furthermore, the composition of the culture medium was sequentially optimized by single-factor experiments, full factorial design, and orthogonal design. Our results showed that the optimal incubation time is 30 h post inoculation; however, the initial pH did not affect the yield of prodiginines as long as it was within 6.0~8.0. Through orthogonal design, the optimal fermentation medium composition was determined as: peptone 11 g/L, yeast extract 1 g/L, soybean oil 5 mL/L and MgCl₂.6H₂O 3 g/L in sea water. Verification tests showed that the yield of prodiginines under optimized conditions reached 13.60 mg/L, representing 2.74 times the yield obtained in Marine Broth 2216 medium. These findings pave the way for cost-effective production of prodiginines from <i>S.ruber </i>MCCC 1K03745^T in the pharmaceutical industry.