Objective To perform a spatial analysis of myocardium uhrastructure and the activity of mitochondrial succinate dehydrogenase in sub-acute Keshan disease. Methods Myocardium samples were collected from the cases with sub-acute Keshan disease and non-myocarditis(control), and their ultrastructure was observed under electron microscope. The density of mitochondrion volume and cristal membrane and its volume were measured by a point-counting method, while mitochondrion volume was estimated by water displacement method, succinate dehydrogenase activity of mitochondrion by iron-copper method in sub-acute Keshan disease and non-myocarditis cases. Results The volume ratio of mitochondrion to the cell on myoeardium [(47.79±6.20)%], the area ratio of mitochondrion to sarcoplasm [(55.06±6.50) %], mitochondrion to myofibrils [(1.43±0,41)%], mitochondrion section area[(0.78±0.15)μm2], and ratio of the lesion of cristal membrane area to the matrix area and mitochondrion volume[(67.14±13.96)%, (44.62±13.44)%]in sub-acute Keshan disease group were obviously higher than those in control [(33.20±7.62)%, (38.07±9.43)%, (0.71±0.33)%, (0.44±0.07)μm2, (14.11± 12.51)%, (9.34±11.28)%; t = 3.75,7.93,6.61,36.40,52.65,37.51, all P < 0.05]. The volume ratio of myofibrils to cell[(34.52±5.12)%]and the area ratio of cristae mitochondria to matrix[(32.43±14.42)%]in sub-acute Keshan disease group was obviously less than those in control [(48.51±4.30)%, (86.04±12.37)%; t = 9.85, 53.46, both P < 0.05)]. Succinate dehydrogenase activity was negative in sub-acute Keshan disease group. Conclusions Myocardium ultrastructure changes in sub-acute Keshan disease including the increase of volume and areas of mitochondria and the damage of the cristal membrane in mitochondria. Succinate dehydrogenase activity is decreased or even disappeared.