Maternal inheritance of mitochondria creates a sex‐specific selective sieve through which mitochondrial mutations harmful to males but not females accumulate and contribute to sexual differences in longevity and disease susceptibility. Because eggs and sperm are under disruptive selection, sperm are predicted to be particularly vulnerable to the genetic load generated by maternal inheritance, yet evidence for mitochondrial involvement in male fertility is limited and controversial. Here, we exploit the coexistence of two divergent mitochondrial haplogroups (A and B2) in a Neotropical arachnid to investigate the role of mitochondria in sperm competition. DNA profiling demonstrated that B2‐carrying males sired more than three times as many offspring in sperm competition experiments than A males, and this B2 competitive advantage cannot be explained by female mitochondrial haplogroup or male nuclear genetic background. RNA‐Seq of testicular tissues implicates differential expression of mitochondrial oxidative phosphorylation (OXPHOS) genes in the B2 competitive advantage, including a 22‐fold upregulation of atp8 in B2 males. Previous comparative genomic analyses have revealed functionally significant amino acid substitutions in differentially expressed genes, indicating that the mitochondrial haplogroups differ not only in expression but also in DNA sequence and protein functioning. However, mitochondrial haplogroup had no effect on sperm number or sperm viability, and, when females were mated to a single male, neither male haplogroup, female haplogroup nor the interaction between male/female haplogroup significantly affected female reproductive success. Our findings therefore suggest that mitochondrial effects on male reproduction may often go undetected in noncompetitive contexts and may prove more important in nature than is currently appreciated. In central Panamanian populations of the harlequin beetle riding pseudoscorpion, Cordylochernes scorpioides, males carrying highly divergent mitochondrial haplogroups (A and B2) differ extensively in the expression of mtDNA genes in testicular tissue. DNA profiling demonstrates that B2‐carrying males sire more than three times as many offspring in two‐male sperm competition experiments than do A males, and this B2 competitive advantage cannot be explained by female mitochondrial haplogroup or male nuclear genetic background. Despite conferring strong fitness benefits on males, the B2 haplogroup remains rare in central Panamá as a consequence of male inability to transmit mitochondria to offspring (mother's curse).