Bacteria use transcriptional regulation to respond to environmental stresses. However,only a few transcription factors regulating drug resistance have been reported. In this study,a transcriptional factor library of Mycobacterium smegmatis was screened on drug plates. A novel transcriptional factor,encoded by the MSMEG_2583 (Ms2583) gene directly affecting M. smegmatis Isoniazid (INH) sensitivity was characterized. Over-expressing Ms2583 caused a significant increase in INH sensitivity of the transformed cells. Results of analyzing structural characteristics of Ms2583 showed that Ms2583 is a TetR family transcription factor. The bacterial one-hybrid assays and electrophoretic mobility shift assays demonstrated that Ms2583 could directly bind to its own promoter with specific interaction.These findings will provide a clue for studying the regulatory mechanism of TetR family transcription factor Ms2583.