Coccinia abyssinica (Lam.) Cogn.] (Cucurbitaceae) is a tuberous neutraceutical plant locally known as Anchote (Oromo language). A micropropagation protocol was developed for C. abyssinica. Seeds were planted ex vitro in a pot that contained different soil mix ratios, and two weeks old seedlings were used as a source of explants. Shoot tip and nodal explants were sterilized and cultured on full MS medium variously supplemented with 6-benzylaminopurine (BAP), indole acetic acid (IAA), naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA) to produce adventitious shoot and rooting. High germination percentage (97%) was obtained from decoated seeds sown on soil mix ratios of 2:1:1 (sand, loam soil, coffee husk, respectively). Sodium hypochlorite at a concentration of 2.0% exposure of 5 min gave high percentages of survived nodal (79.43±0.6) and shoot tip (74.33±0.58) explants. 6-Benzylaminopurine (BAP) (3.0 µM) was found to be an optimum concentration for shoot induction, yielding 80% for nodal and 70% for shoot tip explants. The combination of BAP (3.0 µM) with IAA (0.5 µM) was obtained as optimum concentration yielding 13.4 and 11.03 shoots per explants for nodal and shoot tip respectively for shoot multiplication. Half strength MS medium supplemented with IBA (0.5 µM) and IAA (1.5 µM) yielded more than 90% rooting with optimum root number and length. For acclimatization, sterilized soil mix of 2:1:1 (top forest soil: coffee husk: sand) was optimized yielding 80% on transparent polyethylene plastic tube and 82.2% survived plantlets. This protocol provides a foundation for further studies to generate genetically improved C. abyssinica and related cucurbitaceous species.