Mycobacterium avium subsp. paratuberculosis adapts to the environment via the regulation of genes affecting its envelope's composition. Bacteria grown in milk (in vivo conditions) presented differences in the cell wall-associated lipids and in the expression of genes involved in lipid metabolism (FadE8, FadE6 and MAP1420) and host cell invasion (MAP1203, LprL). A different lipid profile was also observed in the envelope of intracellular bacteria after 1 h of infection. Intracellular bacteria showed up-regulation of a LuxR regulator which controls the envelope's composition by up-regulation of FadE8, MAP1420, MAP1203 and LprL and by down-regulation of pks12, mmpL2 and MAP2594. A LuxR-overexpressing strain with a lipid-deficient envelope phenotype, infected epithelial cells more efficiently than the wild-type bacteria; however, it was not more resistant than the wild-type strain to the action of bactericidal proteins. Here we show that LuxR regulates virulence determinants and is involved in mycobacteria adaptation to the host.