MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel β-strands organized in a coherent domain. Heating to 92 and 112 °C was required to dissociate the MspA tetramer and to unfold the β-sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming β-barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6 M urea at 50 °C was reduced 13- and 30-fold, respectively, although the MspA tetramer and the β-sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are β-sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications.