Saprophytic mycobacteria persist during nutrient limitation by consuming atmospheric hydrogen (H2) via uptake hydrogenases. In Mycobacterium smegmatis, two uptake hydrogenases are upregulated during carbon starvation, with H2 oxidation being most active during stationary phase. However, the regulators controlling hydrogenase expression during starvation remain unresolved. In this study, we demonstrate that the glycerol-inducible regulator GylR regulates the synthesis and activity of the hydrogenase Huc in M. smegmatis. We compared huc gene expression and hydrogenase activity between wild-type and mutant strains lacking gylR expression. Whereas Huc was most active in early stationary phase in the wild-type strain, it was highly active throughout exponential and stationary phase in the gylR deficient strains. Consistently, proteomic data showed a large increase in Huc production in the absence of GylR during both phases. The specific growth rate of wild-type M. smegmatis was sevenfold higher than gylR deficient strains when grown with glycerol, but not other substrates as the sole carbon source, indicating the importance of GylR in glycerol catabolism. However, electrophoretic mobility shift assays (EMSAs) showed the GylR protein does not specifically bind to the huc promoter DNA, suggesting it indirectly controls huc expression via a downstream transcription factor. Altogether, these results suggest that carbon catabolite repression controls trace gas oxidation, providing an elegant way for mycobacteria to hierarchically use available energy sources.