Mitochondrial DNA mutations create variation in the efficiency of the oxidative phosphorylation pathway and therefore cellular energy production. Mildly deleterious mutations may reduce the performance of sperm cells in particular, due to their high energy requirements and low number of mitochondria, yet have little or no effect on the viability of somatic cells or ova. Mutations will be maintained in the population, despite the fitness cost for males, because mtDNA is passed down the female line. We looked for this so-called mother's curse effect in our captive colony of European brown hares. Significantly reduced male reproductive success was detected for a divergent haplotype that could be traced back to hares imported from a remote population. Median reproductive success for these hares was 0.17 compared to 0.49 for the indigenous haplotypes (Wilcoxon rank-sum, P = 0.002). No difference was detected for female reproductive success, nor were we able to find a nuclear DNA component to variation in male fertility. Our data are strong evidence for a mother's curse effect persisting despite multiple crossings over seven generations. These data raise important issues relating to the reproductive fitness of small or intermixing populations and have particular implications for the management of populations for conservation.