Over the last decade, research has demonstrated that soil microbes dominate a range of ecosystems across the globe, with many of these environments providing unfavourable conditions for microbial life to survive. Some aerobic soil bacteria possess the ability to scavenge atmospheric H2, harnessing this trace gas as an energy source to support growth and persistence during oligotrophic conditions. The aerobic saprophyte Mycobacterium smegmatis possesses two differentially expressed, oxygen-tolerant uptake hydrogenases, Huc and Hhy, that facilitate H2 oxidation. H2 oxidation by either hydrogenase yields low potential electrons that can be utilised in the electron transport chain during aerobic respiration. With the ability to scavenge trace levels of atmospheric H2, these specialised metalloenzymes are efficient catalysts of H2 oxidation, and enable M. smegmatis to adapt to resource-limited environments. Although huc is known to be upregulated in response to both organic carbon and oxygen starvation, the complex regulatory network mediating huc expression in response to these conditions remains unknown. In this work, a putative transcriptional activator divergently transcribed upstream of the huc structural operon, MSMEG_2260, was transcriptionally silenced using CRISPR-mediated interference. Wildtype and MSMEG_2260 knockdown strains were exposed to three conditions, carbon limitation, hypoxia and elevated levels of H2, and Huc activity and production was investigated. Using activity-based assays, MSMEG_2260 was found to be critical for Huc activity during both carbon and oxygen starvation. These findings were further supported by proteomics, demonstrating that proteins encoded by the huc operon are significantly less abundant in the MSMEG_2260 knockdown strain in comparison to wildtype M. smegmatis. Furthermore, this research also demonstrates that Huc directly responds to H2 availability, with the abundance of proteins encoded by the huc operon and enzymatic activity of Huc increasing at elevated H2 levels. This work expands the current understanding of hydrogenase expression mechanisms in response to carbon and oxygen deprivation, highlighting MSMEG_2260 as the likely transcriptional activator of Huc hydrogenase expression. More broadly, this data could significantly inform commercial hydrogenase applications, especially with hydrogen fuel cell technology emerging as a promising alternative for generating renewable energy.