The first structure for a member of the DUF3349 (PF11829) family of proteins, Rv0543c from Mycobacterium tuberculosis, has been determined using NMR-based methods and some of its biophysical properties characterized. Rv0543c is a 100 residue, 11.3kDa protein that both size exclusion chromatography and NMR spectroscopy show to be a monomer in solution. The structure of the protein consists of a bundle of five α-helices α1 (M1 - Y16), α2 (P21 - C33), α3 (S37 - G52), α4 (G58 - H65) and α5 (S72 - G87) held together by a largely conserved group of hydrophobic amino acid side chains. Heteronuclear steady-state {(1)H}-(15)N NOE, T(1), and T(2) values are similar through-out the sequence indicating that the backbones of the five helices are in a single motional regime. The thermal stability of Rv0543c, characterized by circular dichroism spectroscopy, indicates that Rv0543c irreversibly unfolds upon heating with an estimated melting temperature of 62.5°C. While the biological function of Rv0543c is still unknown, the presence of DUF3349 proteins predominately in Mycobacterium and Rhodococcus bacterial species suggests that Rv0543 may have a biological function unique to these bacteria, and consequently, may prove to be an attractive drug target to combat tuberculosis.