Spermatogenesis is the intricate and coordinated process by which spermatogonia develop into haploid differentiated spermatozoa. Mitochondria are essential for spermatogenesis, and prohibitin (PHB) is closely associated with mitochondrial structure and function during spermatogenesis. Although PHB has been implicated in spermatogenesis in some taxa, its roles in Opsariichthys bidens have not been determined. In this study, the expression patterns and potential functions of PHB in spermatogenesis in O. bidens were characterized using histological microscopic observations, PCR cloning, real-time quantitative PCR (qPCR), Western blotting (WB) and immunofluorescence (IF). The full-length cDNA of Ob-phb was 1500 bp encoding 271 amino acids. A sequence alignment demonstrated that the PHB protein is conserved among different animals. qPCR revealed that phb mRNA is widely distributed in O. bidens and highly expressed in the testes at stages IV and V. WB revealed that Ob-PHB is located in the mitochondria of testes. IF revealed the colocalization of PHB signals and mitochondria. Signals were detected around nuclei in spermatogonia and spermatocytes, gradually moving to the tail region during spermiogenesis, and finally aggregating in the midpiece. These results indicate that Ob-PHB was expressed in the mitochondria during spermatogenesis. In addition, this study proposed Ob-PHB may participate in the degradation of mitochondria and cell differentiation during spermatogenesis.