[Background] The cutR/cutS two component system plays a significant role in secondary metabolite in Streptomyces. [Objective] The aim of the study is to investigate the function of the cutR/cutS two component system in the production of lomofungin in Streptomyces lomondensis S015. [Methods] HPLC was used to analysis fermentation production, meanwhile accessing quantitative real-time PCR to monitor the levels of gene expression. [Results] HPLC results indicated that the yield of lomofungin in S015ΔcutR and S015ΔcutS reach the total amount of 128.1±26.4 mg/L and 61.8±4.5 mg/L respectively, that is 11.5 and 5.5 times of the yield of wild type. Results of qPCR indicated that in S015ΔcutR mutant the relative gene expression of lomo14, lomo10, lphzB, lphzC, lphzE and lphzG reached respectively 1 151.7±88.8, 110.5±5.8, 129.3±7.7, 380.2±34.6, 348.2±42.1 and 299.8±38.2 times of the wild type, and in S015ΔcutS mutant the relative gene expression of lomo14, lomo10, lphzB, lphzC, lphzE and lphzG were respectively 4.3±0.5, 2.2±0.2, 9.3±0.9, 10.3±0.6, 20.7±1.5 and 20.4±0.8 times. [Conclusion] The study shows that the cutR and cutS negatively regulate several main synthetic genes and side-chain modification genes of lomofungin production in S. lomondensis, thereby reducing the production.