Nematanthus wettsteinii (Fritsch) H. E. Moore is one of the most popular ornamental species in China, and is known for its peculiar “goldfish” shape flower and is usually propagated by cuttings. This study aimed to establish in vitro protocols for N. wettsteinii regeneration. In this study, leaves and shoot-tips were used as explants, and the effects of adventitious shoot induction, rooting medium, and plant growth regulators (PGRs) on regeneration from explants were evaluated. Murashige and Skoog (MS) medium was used as the basic substrate. Among the PGRs tested, when leaves were used as explants, 0.1 mg L⁻¹ α-naphthaleneacetic acid (NAA) and 2.0 mg L⁻¹ thidiazuron (TDZ) induced the largest number of adventitious shoots (22.5 per leaf) with a 93.5% induction rate. When shoot-tips were used as explants, 3.0 mg L⁻¹ benzyl adenine (BA) induced the fewest adventitious shoots (2.6 per shoot-tip), while 0.1 mg L⁻¹ NAA and 2.0 mg L⁻¹ TDZ yielded the largest number of adventitious shoot (20.3 per shoot-tip). The adventitious shoots were then transferred to MS medium containing NAA, BA, and gibberellin A₃ (GA₃) for shoot proliferation and elongation. When half-strength MS medium was supplemented with 0.3 mg L⁻¹ indole-3-butyric acid (IBA), shoot rooting was essentially 100%. After transplanting the plantlets to garden soil mixed with river sand and vermiculite (1:1:1, v/v) substrate, 100% of the plantlets survived after 3 wk. These reliable regeneration protocols constitute a significant advance towards the large-scale propagation and conservation of this famous horticultural species.