Hippophae salicifolia D. Don. is a deciduous, temperate, and dioecious tree of great economic and ecological importance. In this study, an in vitro propagation protocol was established for male and female explants of H. salicifolia. The most suitable explants were selected by screening the various types of explants such as dormant and active axillary buds, apical buds, nodal parts, and leaves. The explants were selected every month throughout the year to test the response, and only dormant and active buds were able to generate a response. Accumulation of phenolics was one of the most significant problems in the in vitro propagation of H. salicifolia, which was overcome by using an antioxidant solution of 1500 mg L−1 each ascorbic acid and citric acid followed by a lower temperature treatment, and pre-culture in 1.2% (w/v) agar supplemented with 0.01% (w/v) myo-inositol and 3% (w/v) sucrose. Active buds proved to be the best explant type to propagate H. salicifolia in vitro, either on Murashige and Skoog (MS) or Lloyd and McCown Woody Plant media supplemented with high cytokinin concentrations. Half- and full-strength MS media were used for rooting the in vitro-grown shoots, and the rooting percentage was higher in the half-strength MS media than in the full strength. There were no significant differences in the responses of male and female explants in various culture conditions. Female plants showed better acclimatization than male plants.