Genetic variation outside of the cell nucleus can affect the phenotype. The cytoplasm is home to the mitochondria, and in arthropods often hosts intracellular bacteria such as Wolbachia. Although numerous studies have implicated epistatic interactions between cytoplasmic and nuclear genetic variation as mediators of phenotypic expression, two questions remain. Firstly, it remains unclear whether outcomes of cyto-nuclear interactions will manifest differently across the sexes, as might be predicted given that cytoplasmic genomes are screened by natural selection only through females as a consequence of their maternal inheritance. Secondly, the relative contribution of mitochondrial genetic variation to other cytoplasmic sources of variation, such as Wolbachia infection, in shaping phenotypic outcomes of cyto-nuclear interactions remains unknown. Here, we address these questions, creating a fully crossed set of replicated cyto-nuclear populations derived from three geographically distinct populations of Drosophila melanogaster, measuring the lifespan of males and females from each population. We observed that cyto-nuclear interactions shape lifespan and that the outcomes of these interactions differ across the sexes. Yet, we found no evidence that placing the cytoplasms from one population alongside the nuclear background of others (generating putative cyto-nuclear mismatches) leads to decreased lifespan in either sex. Although it was difficult to partition mitochondrial from Wolbachia effects, our results suggest at least some of the cytoplasmic genotypic contribution to lifespan was directly mediated by an effect of sequence variation in the mtDNA. Future work should explore the degree to which cyto-nuclear interactions result in sex differences in the expression of other components of organismal life history.