Gentiana cruciata L. (Cross gentian) is a medicinal and ornamental plant, threatened in its natural habitats. The wild root extracts of this species are known to exhibit many curative properties. In the present study, an efficient protocol for in vitro propagation of G. cruciata L. was developed from node culture. A semi-solidified Murashige and Skoog (MS) basal medium supplemented with 2.22 mu M 6-benzyladenine (BA), 2.46 mu M indole-3-butyric acid (IBA) and sucrose (3% w/v) improved the production of multiple shoots directly from nodal segments, providing 3.9 shoots per explants on average. The highest rooting (81.7%) was observed with half-strength MS medium supplemented with 2.46 mu M IBA. Plants with well-developed roots were transferred to pots containing turf/vermiculite mixtures and acclimatized in plant growth chamber conditions. Acclimatized plants showed 100% survival and remained healthy. The content of secondary metabolites in the clones was determined by HPLC, and the presence of gentiopicroside, loganic acid, swertiamarin, and sweroside in the samples was confirmed. Gentiopicroside was found to be the major compound.