Virtually all bacteria possess a peptidoglycan layer that is essential for their growth and survival. The β-lactams, the most widely used class of antibiotics in human history, inhibit D,D-transpeptidases, which catalyze the final step in peptidoglycan biosynthesis. The existence of a second class of transpeptidases, the L,D-transpeptidases, was recently reported. Mycobacterium tuberculosis, an infectious pathogen that causes tuberculosis (TB), is known to possess as many as five proteins with L,D-transpeptidase activity. Here, for the first time, we demonstrate that loss of L,D-transpeptidases 1 and 2 of M. tuberculosis (LdtMt1 and LdtMt2) alters cell surface morphology, shape, size, organization of the intracellular matrix, sorting of some low-molecular-weight proteins that are targeted to the membrane or secreted, cellular physiology, growth, virulence, and resistance of M. tuberculosis to amoxicillin-clavulanate and vancomycin.