The reported analyses were aimed at identification of the mitochondrial proteome features which were associated with cytoplasmic male sterility (CMS) in beets. The set of analyzed accessions included CMS, maintainer and restored lines. Mitochondrial preparations were subjected to blue-native electrophoresis followed either by in-gel activity assays or separation in denaturing conditions. The CMS condition was associated with decreased activity of complex V and enhancement of additional complexes with the ATPase activity. This was accompanied by accumulation of heptamer HSP60, preSATP6 and an increase in the fraction of the free ATP9 oligomer (not bound to complex V). The ATP9 effect was reversed upon fertility restoration.The reported work provides one of very few comprehensive comparisons of the mitochondrial proteomes from cytoplasmic male-sterile (CMS) and male-fertile plants. It shows that in beets the CMS trait is associated with altered functioning of the mitochondrial ATP synthase complex. The presence of CMS-specific ATP synthase subcomplexes resembles deficiencies of this enzyme reported for mammalian cells.