Pig hearts with naturally occurring hypertrophic cardiomyopathy (HCM) were isolated to investigate the effects of mitochondrial deficiency at biochemical and molecular levels. Enzyme activities of mitochondrial-encoded cytochrome c oxidase and NADH dehydrogenase in the HCM hearts (n = 12) were lower than that in the controls (n = 12) by 41 ± 29% (P < 0.01) and 43 ± 21% (P < 0.001), respectively. Additionally, Southern blot analysis was conducted to quantify the relative amount of mitochondrial DNA (mtDNA) from the HCM and controls. The relative amount of mtDNA in the HCM hearts was significantly 57 ± 19% (P < 0.001) lower than that in the controls. Both mitochondrial enzyme deficiency and mtDNA depletion were significantly correlated with the degree of cardiac hypertrophy judged based on the ratio of heart/body weight. In conclusion, our results reveal that a secondary effect of tissue-specific mtDNA depletion and mitochondrial dysfunction is in response to the HCM.