D-xylose is the most abundant fermentable pentose, which usually represents an architectural component of the bacterial cell wall. However, its regulatory function and the involved signaling pathway in bacteria remain largely unclear. Here, we show that D-xylose can act as a signaling molecule to regulate the lipid metabolism and affect multiple physiological characteristics in mycobacteria. D-xylose directly interacts with XylR and inhibits its DNA-binding ability, thus blocking XylR-mediated repression. The xylose inhibitor, XylR, plays a global regulatory role and affects the expression of 166 mycobacterial genes that are involved in lipid synthesis and metabolism. Furthermore, we show that the xylose-dependent gene regulation of XylR affects the multiple physiological characteristics of , including bacterial size, colony phenotype, biofilm formation, cell aggregation, and antibiotic resistance. Finally, we found that XylR inhibited the survival of BCG in the host. Our findings provide novel insights into the molecular mechanism of lipid metabolism regulation and its correlation with bacterial physiological phenotypes.