The primary aim of the present study was to examine the effect of maternal age (in months) on mitochondrial DNA copy number (Mt number), ATP content and IVF outcome of bovine oocytes. We also compared the Mt number of oocytes with fertilisation outcome and ATP content. Oocytes were collected from cows aged 20-204 months and the Mt number was determined by real-time polymerase chain reaction. The Mt number in immature and mature oocytes was determined to be 368 118 and 807 794, respectively; the ATP content in these oocytes was 1.2 and 2.0 pM, respectively. Both Mt number and ATP content increased during oocyte maturation. However, after 90 months of age, the Mt number of mature oocytes decreased with increasing maternal age, whereas the ATP content of mature oocytes was positively correlated with maternal age (P<0.01); there was no obvious relationship observed between Mt number and ATP content. Furthermore, maternal age was positively correlated with the abnormal fertilisation rate (P<0.01). Mt number and fertilisation outcome were unrelated, but the nature of this relationship differed between young (21-89 months) and old (>89 months) cows. Thus, we conclude that Mt number, the ATP content and fertilisation outcome of bovine oocytes are affected by maternal age.