<p><i>Gynura divaricata</i> (L.) DC., a herb with economic, ecological, and medical importance, is widely cultivated in tropical and subtropical areas of Southeast Asia. <i>G. divaricata</i> can bloom and set seeds, but only a small number of seeds form and very few are viable, so seedlings are rarely employed for propagation purposes. In this study, callus and adventitious shoots were induced from leaves. Adventitious shoot propagation is reported for the first time for this species. The optimal medium for direct induction of adventitious shoots from leaf explants was Murashige and Skoog (MS) medium containing 4.0 mg L^− 1 thidiazuron (TDZ), forming 7.78 adventitious shoot buds per explant. When 2.0 mg L^− 1 TDZ and 0.5 mg L^− 1 α-naphthaleneacetic acid (NAA) or 2.0 mg L^− 1 6-benzyladenine (BA) and 0.5 mg L^− 1 NAA were added to MS medium, callus was induced initially, followed by 9.78 or 8.87 adventitious shoot buds (per explant), which developed from callus. Adventitious shoots could not be induced by 2,4-dichlorophenoxyacetic acid, NAA or kinetin. The addition of L-ascorbic acid (AsA) to MS medium reduced the level of hyperhydricity of shoots and plantlets. The optimal medium for shoot proliferation was MS medium with 4.0 mg L^− 1 BA and 50 mg L^− 1 AsA. Optimal rooting of <i>G. divaricata</i> was achieved on MS medium with 0.5 mg L^− 1 indole-3-butyric acid, with 97.33% of shoots forming roots within 1 week. The optimal substrate for transplantation was a mixture of peat and vermiculite or peat and fine sand (1:1, <i>v/v</i>). After 3 weeks, the survival percentage of transplanted plantlets was 98.15%.</p>