Extractable glycolipids of mycobacteria, such as lipooligosaccharides (LOSs), play crucial roles in responding to environmental stress and modulating the host immune response. Although the biosynthesis of LOS is likely regulated at multiple levels to ensure proper composition of the cell wall, the key regulators remain unknown. In this study, we investigated B11, a conserved mycobacterial small RNA (sRNA), and found that it post-transcriptionally regulates LOS synthesis in . Through a combination of RNA-seq and mass spectrometry screening, we identified specific genes within the LOS synthesis locus that are directly regulated by B11. We confirmed in vivo sRNA-mRNA interactions using MS2-tagged RNA affinity purification, and found that B11 utilizes the cytosine-rich loop of its Rho-independent transcriptional terminator to interact with guanine tracks adjacent to the ribosome binding sites of its target genes, thereby impeding translation and promoting mRNA degradation. Moreover, deletion of B11 altered the colony morphology associated with LOS composition. These comprehensive functional studies of the mycobacterial sRNA B11 reveal sRNA-based regulation of LOS synthesis, providing new insights into the regulatory mechanisms controlling the biosynthesis of the complex mycobacterial cell wall.