Mycolic acids, the major lipid of the Mycobacterium tuberculosis cell wall, are modified by cyclopropane rings, methyl branches, and oxygenation through the action of eight S-adenosylmethionine (SAM)-dependent mycolic acid methyltransferases (MAMTs), encoded at four genetic loci. Mycolic acid modification has been shown to be important for M. tuberculosis pathogenesis, in part through effects on the inflammatory activity of trehalose dimycolate (cord factor). Studies using the MAMT inhibitor dioctylamine have suggested that the MAMT enzyme class is essential for M. tuberculosis viability. However, it is unknown whether a cyclopropane-deficient strain of M. tuberculosis would be viable and what the effect of cyclopropane deficiency on virulence would be. We addressed these questions by creating and characterizing M. tuberculosis strains lacking all functional MAMTs. Our results show that M. tuberculosis is viable either without cyclopropanation or without cyclopropanation and any oxygenated mycolates. Characterization of these strains revealed that MAMTs are required for acid fastness and resistance to detergent stress. Complete lack of cyclopropanation confers severe attenuation during the first week after aerosol infection of the mouse, whereas complete loss of MAMTs confers attenuation in the second week of infection. Characterization of immune responses to the cyclopropane- and MAMT-deficient strains indicated that the net effect of mycolate cyclopropanation is to dampen host immunity. Taken together, our findings establish the immunomodulatory function of the mycolic acid modification pathway in pathogenesis and buttress this enzyme class as an attractive target for antimycobacterial drug development.