Ephedra foliata Boiss. ex C.A. Mey (Ephedraceae) is a well-known source of the alkaloid ephedrine used for the treatment of chronic asthma and associated respiratory ailments. This investigation was conducted to standardize an efficient regeneration protocol for E. foliata via indirect organogenesis through intermodal explants. Maximum callus induction (80.3%) was achieved on Murashige and Skoog (MS) medium supplemented with 0.5 mg L‑1 2,4‑dichlorophenoxyacetic acid + 0.5 mg L‑1 kinetin and additives (50 mg L‑1 ascorbic acid and 25 mg L‑1 citric acid). The maximum shoot regeneration (n = 5.27) was achieved on MS medium containing 6.0 mg L‑1 kinetin, followed by 5.0 mg L‑1 6‑benzylamino purine (n = 4.27). MS half strength medium with 3.0 mg L‑1 α‑naphthalene acetic acid resulted in the highest rooting percentage (32%). Sixty percent of the plantlets survived during acclimatization and were successfully transferred under field conditions. These plants and callus will be a suitable source of plant material for mass multiplication, genetic modification for enhanced bioactive constituents, and germplasm conservation.