Milk synthesis by the mammary gland declines during prolonged lactation despite the continued suckling stimulus and complete removal of mammary secretions. Although this process has been hypothesized to result from cellular aging there has been no reported analysis of aging markers in the lactating mammary gland. The goal of these studies was to relate lactation performance in the mouse during a single prolonged lactation cycle to changes in mammary development and mitochondrial oxidative damage. During an artificially prolonged lactation cycle, the capacity of the dams to support litter growth decreased over time. This decrease was associated with decreased mammary epithelial content. Cell proliferation, along with the percentage of mammary progenitor cells, was high during early lactation, but low during prolonged lactation. Apoptosis increased during prolonged lactation. Oxidative damage to mitochondrial DNA increased during the early postpartum period and remained elevated through the end of the cycle. In contrast oxidative damage to mitochondrial protein was high during early lactation and decreased through mid lactation to increase again with prolonged lactation. The results suggest that a single prolonged lactation cycle may replicate on an accelerated basis some of the changes that occur with a lifetime of aging in organs possessing more stable cell populations.