New marine bacterium sp. SY01, producer of prodigiosin, was isolated from the seawaters of Sanya Bay. The culture conditions of this bacterium were investigated. sp. SY01 was cultured in 2216E media which contained tryptophan, histidine, lactonic acid, camphor, limonene, casein, diphenyl guanidine, coumarin and 1,3-dinitrobenzene, respectively. After 5 days cultivation, the extracts of different culture broths were detected by direct infusion mass spectroscopy using positive ESI mode. As the results, tryptophan, histidine and casein didn’t show any observable influences on the biosynthesis of prodigiosin. Lactonic acid, camphor, limonene, diphenyl guanidine, coumarin could inhibit the bacterium growth and prodigiosin biosynthesis to a certain extent, slower the culture broth to turn red. However, 1, 3-dinitrobenzene inhibited the bacteria to produce prodigiosin completely. MS data suggested that various metabolites with chemodiversity were produced in different culture media. In particular, a series of high-molecular-weight compounds with high relative abundances were observed in the medium containing limonene. To further optimize the culture condition, more new prodigiosin analogues and lead compounds can be obtained and the goal of “one strain-many compounds” can be achieved.