Carotenoids are implicated in alleviating ageing and age-related diseases in humans. While data from different carotenoids are mixed in their outcomes, those for 9-cis-β-carotene indicate general positive effects, although basic data on its biological impact are limited. Here, we show that supplementation with 9-cis-β-carotene in ageing Drosophila melanogaster improved mitochondrial function in terms of ATP production and whole-body respiration and extended mean lifespan. It also resulted in improved mobility. These data provide a potential biological rational for the beneficial effects of dietary supplementation with 9-cis-β-carotene. These effects may be based on the maintenance of a sound mitochondrial function.