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A novel method for producing high-quality lycopene from Blakeslea trispora through submerged fermentation is studied. 2-Isopropylimidazole at 300 mg/L completely inhibited lycopene cyclase, which limited the accumulation of γ-carotene and β-carotene. 2-Isopropylimidazole also intensified the metabolic flow of lycopene by increasing the expression levels of key enzymes involved in the lycopene synthetic pathway. Ketoconazole at 20 mg/L reduced the metabolic flow of ergosterol and increased lycopene content and production by 76.6% and 64.3%, respectively. Lycopene accounted for approximately 96% of the total carotenoids, whereas cis-lycopene accounted for approximately 35% of the total lycopene. The content and nutritional quality of lycopene from B. trispora were higher than those of lycopene from tomatoes. B. trispora is an ideal source of natural lycopene.