Clustered regularly interspaced short palindromic repeat interference (CRISPRi) is a new type of transcriptional repression technology. The system contains RNA-mediated DNA endonuclease dCas9 and specific single guide RNA (sgRNA) for the target gene. The DNA recognition complex recognizes the corresponding DNA sequence to suppress transcription of the target gene. Isocitrate dehydrogenase (ICD) is a key metabolic enzyme in the tricarboxylic acid cycle (TAC) and plays an important role in the carbon metabolism of Mycobacteria. In this study, icd knockdown (ICD-KD) strain of Mycobacterium smegmatis (M. smegmatis) was constructed by using CRISPRi to efficiently inhibit the expression of mycobacterial specific genes. The results of quantitative polymerase chain reaction (qPCR) and Western blotting showed the significant decrease in the transcriptional level and protein expression level of ICD, indicating that M. smegmatis ICD-KD strain was constructed successfully by using CRISPRi technique. The growth rate of ICD-KD strain decreased significantly. At the same time, enzyme activity of ICD decreased significantly. It is suggested that CRISPRi technology could rapidly construct knockdown strains of essential mycobacterial genes, laying an important foundation for the subsequent study of mycobacterial ICD function in the carbon source metabolic pathway and carbon flux flow in the TAC.