Mitochondria are essential for spermiogenesis. Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins that act as scaffolds in the inner mitochondrial membrane. In this study, we analyzed the molecular structure and dynamic expression characteristics ofOt-PHBs, observed the colocalization ofOt-PHB1 with mitochondria and polyubiquitin, and studied the effect ofphb1knockdown on mitochondrial DNA (mtDNA) content, reactive oxygen species (ROS) levels, and apoptosis-related gene expression in spermatids. Our aim was to explore the effect ofOt-PHBs on mitochondrial function during the spermiogenesis ofOctopus tankahkeei(O. tankahkeei), an economically important species in China. The predictedOt-PHB1/PHB2 proteins contained an N-terminal transmembrane, a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin domain), and a C-terminal coiled-coil domain.Ot-phb1/phb2mRNA were widely expressed in the different tissues, with elevated expression in the testis. Further,Ot-PHB1 andOt-PHB2 were highly colocalized, suggesting that they may function primarily as anOt-PHB compiex inO. tankahkeei.Ot-PHB1 proteins were mainly expressed and localized in mitochondria during spermiogenesis, implying that their function may be localized to the mitochondria. In addition,Ot-PHB1 was colocalized with polyubiquitin during spermiogenesis, suggesting that it may be a polyubiquitin substrate that regulates mitochondrial ubiquitination during spermiogenesis to ensure mitochondrial quality. To further investigate the effect ofOt-PHBs on mitochondrial function, we knocked downOt-phb1and observed a decrease in mtDNA content, along with increases in ROS levels and the expressions of mitochondria-induced apoptosis-related genesbax,bcl2, andcaspase-3mRNA. These findings indicate that PHBs might influence mitochondrial function by maintaining mtDNA content and stabilizing ROS levels; in addition, PHBs might affect spermatocyte survival by regulating mitochondria-induced apoptosis during spermiogenesis inO. tankahkeei.