Micromonospora carbonacea JXNU-1, a strain of an actinomycete with broad-spectrum antimicrobial activity, isolated from soil samples from the farmland in the area of Yaohu Lake in Nanchang, China, was taken as the object of study in this paper. Bioinformatics analysis revealed that there was a pair of proteins MCNtrB/MCNtrC homologous to the two-component system NtrB/NtrC, which usually exists only in the Gram-negative bacteria and is closely related to the regulation of nitrogen metabolism, in the whole cell protein of M. carbonacea. MCNtrB and MCNtrC, obtained by expression in vitro, were subjected to autophosphorylation and phosphate group transfer experiments. The results showed that MCNtrB had histidine kinase activity with the active site of His115, and MCNtrC can accept the phosphate group from phosphorylated MCNtrB with the active site of Asp33. The yeast two-hybrid experiments showed that MCNtrB and MCNtrC were a pair of proteins with a strong interaction. Overexpression of MCNtrB and MCNtrC in M. carbonacea can affect the expression of key enzymes in cellular nitrogen metabolism, such as glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. These results indicated that MCNtrB/MCNtrC is a two-component system related to nitrogen metabolism in M. carbonacea, which could provide an important experimental basis for revealing the regulatory mechanism of nitrogen metabolism in M. carbonacea.