Cucumis hystrix Chakr. is a rare cucurbit species native to Asia. The species is valued by breeders because of its multiple branching habit and has been used in interspecific crosses with Cucumis sativus. However, individual C. hystrix plants have not been identified in the wild since 1990. Therefore, it was our objective to develop a micropropagation protocol that would allow us to clonally propagate plants in cultivation. Shoots tips (2 cm) were excised from a single C. hystrix plant grown in the greenhouse. All tendrils and leaves were removed before surface-sterilization in 1.25% NaOCl for 5 or 10 min and rinsed six times with sterile distilled water. Shoot tips were trimmed to 1 cm (meristem with two to three young leaf primordia) and placed into 25 × 125-mm test tubes containing 25 ml of initiation medium [MS plus (per liter) 100 mg inositol, 30 g sucrose and 5 g Agargel; pH 5.7-5.8]. PGR combinations tested were initiation medium with 1 μM BA, and initiation medium with 1.7 μM IBA, 0.5 μM kinetin and 0.3 μM GA3 (IKG). Explant survival was greater when shoot tips were surface-sterilized for 5 min (75%) compared to 10 min (33%). More axillary shoots formed when shoot tips were cultured in IKG medium (10.8) than in medium with BA (5.5). Shoots were considerably longer (10 mm) when cultured in medium with IKG compared to BA (1.5 mm). About 64% of shoots place in medium containing 8 μM NAA formed roots and were acclimatized to greenhouse conditions.