The roots of Pyrenacantha volubilis contain camptothecin (CPT), a high-value bioactive compound possessing anticancer and anti-HIV properties. Isolated root cultures of P. volubilis established in half MS media fortified with 0.3 mgL-1 indole-3-acetic acid and 0.2 mgL-1 indole-3-butyric acid and transferred to light conditions resulted in induction of green roots which obtained a maximum biomass content of 1.09 ± 0.03 g fresh weight with a growth index of 2.07 ± 0.02 in 60 days. The chlorophyll and camptothecin content of the roots was found to increase throughout the culture period attaining a maximum total chlorophyll content of 2.97 ± 0.004 mgg-1 fresh weight in 60 days and CPT content of 2.59 ± 0.02 mgg-1dry weight in 50 days. In the elicitation studies, the green roots treated with 200 mgL-1 yeast extract yielded a maximum camptothecin content of 5.3 ± 0.40 mgg-1 dry weight in 7 days of incubation, which was a 1.94 fold increase in camptothecin content than the control cultures. The thin layer chromatography, high performance liquid chromatography, and liquid chromatography-mass spectroscopy analysis further confirmed the presence of camptothecin (2.17 ± 0.04 mgg-1DW) in the 30 day old in vitro root samples. The overall results suggest the feasibility of green root cultures of P. volubilis as an efficient system for sustainable in vitro production of camptothecin provided further scaling up experiments in bio reactors are imperative.