The mitochondrion is an important cellular component responsible for regulating energy, oxidative metabolism, and acclimatization to high altitude. This study is aimed at investigating the impact of long-term exposure to hypoxia on the amount of mitochondrial DNA (mtDNA) in rat livers. Male Sprague-Dawley rats were randomized and exposed to normoxia (group I), or 5,000 m (barometric pressure about 405.35 mmHg) above the sea level (a hypoxic condition) for 23 and 1 h normoxia daily for five consecutive days (group II), 15 days (group III), and 30 days (group IV), respectively. The levels of plasma malondialdehyde (MDA), homocysteine (Hcy), superoxide dismutase (SOD), and alanine aminotransferase (ALT), the contents of liver mtDNA, mitochondrial transcription factor A (mtTFA), cytochrome oxidase 1 (COX1), COX2, and COX3 mRNA transcripts, and mitochondrial respiratory activity were examined immediately after the last cycle. In comparison with that in control rats, 5-15 cycles of hypoxia/normoxia significantly increased the levels of plasma MDA and ALT, but reduced the levels of Hcy and SOD, accompanied by impairing liver respiratory function in rats. Long-term (30) cycles of hypoxia/normoxia reduced the levels of plasma MDA and ALT, but increased the levels of SOD and Hcy, accompanied by decreased mtTFA expression and mtDNA amount, improved mitochondrial respiratory function in rat liver, when compared that of 5-15 cycles of hypoxia/normoxia. Our data indicate that long-term cycles of hypoxia/normoxia increases the amount of mtDNA and up-regulates COX expression, contributing to acclimatization to very high altitude in rats.