Induction mechanism of a potential red pigment (RPc) was investigated in the present paper. A typical competition relationship exists between Penicillium sp. HSD07B and Candida tropicalis during co-culture, and C. tropicalis converts glucose into glycerol, organic acids and other substances, resulting in a stricter glucose limitation and the secretion of RPc. Moreover, a novel eutrophic-oligotrophic transition cultivation system (E-OTCS) was developed to produce red pigment during monoculture of Penicillium sp. HSD07B. However, the monoculture pigment (RPm) is different from RPc in components, and RP3 and RP4 only occur in RPm when glycerol is supplied. In addition, the additions of glycerol and organic acids to glucose exhaustion medium can significantly improve the pigment yield. These facts not only prove the feasibility of producing RPm using E-OTCS, but also reveal that, besides glucose exhaustion, the accumulation of metabolites of glucose including glycerol and organic acids is also an important factor influencing the production of RPc.