The soil bacterium Mycobacterium smegmatis survives during carbon starvation by oxidising atmospheric H2. However, the regulators that control the switch from a carbon-based growth to H2-based survival are unknown. Here, we isolated several transcription factors that potentially regulate the H2-oxidising hydrogenase, Huc. To identify transcriptional regulators of the huc operon, we used a DNA pulldown assay with the huc promoter as bait. We incubated huc promoter DNA with cell lysate from an M. smegmatis strain lacking the glycerol-responsive transcriptional regulator gylR, which exhibits increased Huc activity due to a dramatic upregulation of huc expression. The DNA pulldown was coupled with gel electrophoresis and mass spectrometry, which identified five transcriptional regulators that bound the huc promoter DNA. Subsequent electrophoretic mobility shift assays (EMSA), using purified huc promoter DNA and transcriptional regulator proteins, were carried out to confirm the interactions. To understand the possible role of these transcription factors in the activation or repression of huc expression, we used CRISPRi technology, to knock them down in M. smegmatis gylR mutant and wild-type strains. Altogether, these findings suggest a novel regulator could control metabolic adaptation of mycobacteria to starvation.