Both pathogenic and non-pathogenic Mycobacteria spread across surfaces using sliding motility. Sliding motility is a passive form of bacterial motility in which cells use the expansive force of cell growth usually aided by a secreted surfactant to spread as a monolayer. In Mycobacteria, the components of the mycobacterial outer membrane are involved in sliding motility, including glycopeptidolipids (GPLs) in Mycobacterium smegmatis and lipooligosaccharides (LOSs) in Mycobacterium marinum. Lsr2, an H-NS-like nucleoid-associated protein, negatively regulates sliding motility in M. smegmatis, but the mechanism by which it regulates sliding motility is not known. In a Δlsr2 strain 162 genes show increased transcript levels including the LOS synthesis genes (1). We hypothesized that the increase in transcript levels of the LOS synthesis genes is responsible for the Δlsr2 strain’s hypermotile phenotype. We deleted the gene MSMEG_4727, which is required for LOS synthesis, in both wild-type M. smegmatis and Δlsr2 backgrounds. While the ΔMSMEG_4727 strain showed no change in motility, the Δlsr2 ΔMSMEG_4727 strain showed a decrease in sliding motility, although not down to wild-type levels. This shows that the LOSs are partially responsible for the increased motility of the Δlsr2 strain, but that other factors are likely involved. We also deleted the mps1 gene, required for GPL synthesis, and showed that GPLs are required for motility in the Δlsr2 strains, indicating that the change in biofilm formation and sliding motility in the Δlsr2 strain are through different mechanisms. A screen for new hypermotile mutants yielded two mutants with new phenotypes, although the location of the mutations is not yet know. We also showed that M. smegmatis produces a surfactant in sliding conditions using an atomized oil assay. The surfactant is produced at low levels in the mc²155 lab strain and takes over 10 days to be visible, but at a higher level in the ancestral strain ATCC607. Future work identifying the surfactant and further characterizing the new hypermotile mutants will provide a more clear understanding of sliding motility in M. smegmatis.