Isoëtes sabatina is an aquatic quillwort endemic to Italy. It is one of the rarest quillworts in Europe, and is critically endangered due to restricted range and to the continuous decline of both population and habitat quality. This study aims to develop an optimized protocol to reproduce and grow I. sabatina sporelings. Mature and immature megaspores were mixed with mature microspores to evaluate the influence of the developmental stage on germination and sporeling development. Two substrates, distilled water and water-agar medium, were tested for germination and sporeling emergence, and three substrates, sand, lake sediment and water-agar, were tested for transplants. A high percentage of megaspore germination (a total of 79.1%) was obtained in both substrates, higher for mature than immature spores. A total of 351 sporelings were produced in distilled water and water-agar cultures, with similar percentages (64.5% and 69.6%, respectively). The development stage of the megaspores affected both germination and sporeling development. Sporeling emergence showed significantly higher percentages in mature megaspores than immature ones (69.6% vs. 11.6%, respectively), with 85% of germinated spores developing sporelings. Only transplants over water-agar medium were successful. This protocol could be useful for the propagation of sporelings as the key step towards the planning of in situ actions to save this Mediterranean quillwort from extinction.