The present invention provides live, attenuated Mycoplasma gallisepticum bacteria that exhibit reduced expression of a protein identified as MGA_0621. In certain embodiments, the attenuated bacteria may additionally exhibit reduced expression of one or more proteins selected from the group consisting of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35, relative to a wild-type M. gallisepticum bacterium. Also provided are vaccines and vaccination methods involving the use of the live, attenuated M. gallisepticum bacteria, and methods for making live attenuated M. gallisepticum bacteria. An exemplary live, attenuated strain of M. gallisepticum is provided, designated MGx+47, which was shown by proteomics analysis to exhibit significantly reduced expression of MGA_0621, and was shown to be safe and effective when administered as a vaccine against M. gallisepticum infection in chickens.