Mitochondrial homeostasis plays a pivotal role in oocyte maturation and embryonic development. Deoxyguanosine kinase (DGUOK) is a nucleoside kinase that salvages purine nucleosides in mitochondria and is critical for mitochondrial DNA replication and homeostasis in non-proliferating cells. Dguok loss-of-function mutations and deletions lead to hepatocerebral mitochondrial DNA deletion syndrome. However, its potential role in reproduction remains largely unknown. In this study, we find that Dguok knockout results in female infertility. Mechanistically, DGUOK deficiency hinders ovarian development and oocyte maturation. Moreover, DGUOK deficiency in oocytes causes a significant reduction in mitochondrial DNA copy number and abnormal mitochondrial dynamics and impairs germinal vesicle breakdown. Only few DGUOK-deficient oocytes can extrude their first polar body during in vitro maturation, and these oocytes exhibit irregular chromosome arrangements and different spindle lengths. In addition, DGUOK deficiency elevates reactive oxygen species levels and accelerates oocyte apoptosis. Our findings reveal novel physiological roles for the mitochondrial nucleoside salvage pathway in oocyte maturation and implicate DGUOK as a potential marker for the diagnosis of female infertility.