Micropropagation of Dianthus gigantheus D’Urv. ssp. croaticus (Borbás) Tutin, a Croatian neoendemic plant species, was investigated. Shoots from aseptically germinated seeds were used for culture initiation. The highest multiplication rate (3.3 shoots per explant) was achieved on basal MS medium containing 2.9 µM gibberellic acid and 0.5 µM 6-benzylaminopurine. The rooting percentage was high on all media tested (basal MS medium supplemented with different concentrations of indole-3-acetic acid, indole-3-butyric acid and α-naphthalene acetic acid or without them), with slight suppression on media containing higher concentrations of α-naphthalene acetic acid. Plantlets were successfully acclimatized to outdoor conditions.