Elasmobranchs have an ancestral reproductive system, which offers insights into vertebrate reproductive evolution. Despite their unchanged design over 400 million years, they evolved complex mechanisms ensuring reproductive success. However, human activities induced a significant decline in elasmobranch populations worldwide. In the Mediterranean basin, the smooth-hound shark (Mustelus mustelus) is one of the species that are considered vulnerable to human activities. Conservation efforts necessitate a thorough understanding of its reproductive strategy. This study focused on mature male specimens of smooth-hound sharks that were captured in the Adriatic area and successively analyzed to provide, for the first time, a histologically detailed description of testicular development in the species. Seven phases of the spermatogenesis process were identified, along with the macromolecular characterization of cells obtained using Fourier-transform infrared imaging. Histological analysis showed structural and cellular features similar to those documented in the spermatocysts of other elasmobranchs. The examination of the evolution and migration of both germinative and Sertoli cells at each phase revealed their close connection. Furthermore, different expression levels of lipids, proteins, and phosphates (DNA) at each spermatogenesis stage were observed. This research provided new information on spermatogenesis in the common smooth-hound shark, which is crucial for conservation efforts against population decline and anthropogenic pressures.