Rumohra adiantiformis, also known as “leatherleaf fern”, is an ornamental species that, because of its long display life, is widely used in floral arrangements. In this study, a new protocol for in vitro regeneration of the leatherleaf fern was established. For spore germination, two culture media (MS and Knop) were assessed with presence or absence of 1g L⁻¹ activated charcoal (AC) under different light and dark conditions. Frond, frond microcuttings, and prothallus explants were evaluated on Knop regeneration medium supplemented with 1g L⁻¹ AC, 0.5% agar, pH 5.0 in combination with 2,4 dichlorophenoxyacetic acid (2,4-D: 0.0, 0.1, 0.5 and 1.0 mg L⁻¹) and 6-benzylamino purine (BA: 0.0, 0.1, 0.5 and 1.0 mg L⁻¹). For rooting, four levels of α-naphthaleneacetic acid (NAA: 0.0, 0.01, 0.1 and 0.2 mg L⁻¹) were tested. After 18 days of culture, spore germination rate was 100% on Knop medium with AC and 8 h light/16 h dark. After 120 days of culture, sporophytes 1.7 ± 0.4 cm in length developed on Knop medium, while those spore-cultured on MS medium never produced sporophytes. From those germinated sporophytes, prothallus explants cultivated on Knop medium with AC and 0.5 mg L⁻¹ BA showed the highest regeneration rate with 235.7 gametophytes. The best sporophyte rooting response was obtained with 0.01 mg L⁻¹ NAA. Complete, regenerated sporophytes were obtained 183 days after culture initiation. By this procedure it would be possible to obtain up to 2 million sporophytes from one fertile frond. To determine the origin of the regenerated gametophytes, a histological analysis was performed with scanning electron microscopy (SEM). The analysis revealed that the gametophytes were regenerated from explant epidermal tissues on either the adaxial or abaxial surface.