Recombination-based tools for introducing targeted genomic mutations in Mycobacterium tuberculosis are not efficient due to higher rate of illegitimate recombination compared with homologous DNA exchange. Moreover, involvement of multiple steps and specialized reagents make these tools cost ineffective. Here we introduce a novel clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) approach that efficiently represses expression of target genes in mycobacteria. CRISPRi system involves co-expression of the catalytically dead form of RNA-guided DNA endonuclease from the type II CRISPR system known as dCas9 and the small guide RNA specific to a target sequence, resulting in the DNA recognition complex that interferes with the transcription of corresponding DNA sequence. We show that co-expression of the codon-optimized dCas9 of S. pyogenes with sequence-specific guide RNA results in complete repression of individual or multiple targets in mycobacteria. CRISPRi thus offers a simple, rapid and cost-effective tool for selective control of gene expression in mycobacteria.