The tissue culture and rapid propagation of Cleome spinosa Jacq. was explored by investigating the effects of different plant growth regulators on callus induction, bud differentiation, and root formation of three types of explants. The results showed that hypocotyls and stem segments regenerated buds directly on growth regulator-free Murashige and Skoog (MS)medium. The highest callus induction rates of hypocotyls, stem segments, and leaves reached 100% and were obtained on the culture medium of MS + (1.0 to 2.0) mg/L kinetin (KT) + 0.02 mg/L α-naphthalene acetic acid (NAA), on which the leaves produced the best quality of calluses. The leaf-derived calluses were subcultured on MS + 0.5 mg/L KT +0.5 mg/L 6-benzylaminopurine (BAP) and achieved the highest differentiation rate of 100%, producing an average of 7.5 buds per explant. Inoculation with MS + 0.5 mg/L indole-3-butyric acid (IBA) resulted in the production of a number of thick roots by 66.7% of the regenerated buds. After transplanting, plantlets with more roots survived easily and grew well.