As part of conservation efforts for Pinna nobilis, a critically endangered bivalve endemic to the Mediterranean Sea, laboratory programs have been developed to maintain and breed specimens. However, progress in the ex situ conservation of the species remains limited and challenging. This study aims to advance the knowledge required to establish effective reproductive protocols for P. nobilis, specifically focusing on the population in the Mar Menor lagoon, one of the last two surviving populations along the Spanish coast. The first phase of this study involved characterizing the reproductive events in the lagoon. Subsequently, two ex situ reproduction experiences were conducted under conditions designed to replicate the lagoon's natural environment. Three reproductive events were detected in the lagoon between 2019 and 2022, and five successful spawning events occurred across the two ex situ experiences. The conditions for maintenance, maturation, and induction of the individuals are described. In all cases, the percentage of fertilized oocytes released was remarkably high, suggesting internal fertilization, but not self-fertilization, within the pallial cavity. Additionally, ex situ individuals exhibited simultaneous hermaphroditism, with synchronous maturation and alternating release of gametes, effectively preventing self-fertilization. These findings represent a significant step forward in understanding the reproductive biology of P. nobilis and contribute to efforts aimed at ensuring the species’ long-term survival.