Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), one of the deadliest diseases worldwide. The emergence of multidrug-resistant (MDR) TB and extensive drug-resistant (XDR) TB is a large challenge to TB treatment. In this study, a transcriptional factor library of Mycobacterium smegmatis as a model strain for pathogenic M. tuberculosis was screened on plates containing 15 μg/mL Isoniazid (INH).A novel transcriptional factor encoded by MSMEG_3469 (Ms3469) directly affecting M. smegmatis INH sensitity was characterized. Over-expression of Ms3469 caused a significant increase in INH resistance of M. smegmatis. Results of bioinformatics analyses showed that Ms3469 is a member of the TetR/AcrR family of transcription regulators and forms a homodimer similar to SO_TetR.The electrophoretic mobility shift assays demonstrated that Ms3469 could bind to its own promoter.The direct and specific interaction was further confirmed by bacterial one-hybrid assays.It will provide a clue for studying the regulatory mechanism of bacterial drug resistance in M. smegmatis.