<p>This research focused on the importance of the in vitro culture for rapid multiplication of <i>Calotropis procera</i>, a local herb of great medicinal value in Iran. It also looked at the employment of a suitable elicitor for increasing its growth and therapeutic substances production. In this regard, initially, optimizing the culture medium for in vitro culture was performed. Next, it was assessed how multi-walled carbon nanotubes (MWCNTs) help improve growth traits, physiological parameters, element uptake, and secondary metabolites of <i>C. procera</i>. Here, quarter-strength MS basal medium was considered as an optimal medium for in vitro culture of <i>C. procera</i>. Studying the impact of different amounts of MWCNTs (0, 100, 200 and 400 mg L⁻¹) on the growth and physiological parameters showed a dose-dependent effect of MWCNTs. After 4 weeks of treatment, growth of seedlings, their chlorophyll contents, antioxidant compounds, and the uptake of essential nutrients incresed at 200 mg L⁻¹ MWCNTs, and then a decline was observed at 400 mg L⁻¹. Increased activities of superoxide dismutase and ascorbate peroxidase, and carotenoids accompanied with lower amounts of essential elements at 400 mg L⁻¹ MWCNTs suggested that the high concentration of MWCNTs might induce oxidative stress. Additionally, 200 mg L⁻¹ MWCNTs greatly increased the amounts of phytochemical markers in <i>C. procera</i> seedlings including camphor, linalool, p-cymene, germacrene D, citronellal, geranial, and citronellol. Generally, these results solidify the claim that MWCNTs treatment under in vitro cultures is a good alternative approach for increasing the biomass and biosynthesis of antioxidants in <i>C. procera</i>.</p>