Helicteres isora is a medicinal plant effective against asthma, diabetes, hypolipidemia, HIV, polio besides a good source of diosgenin. Seed dormancy and low natural fruit production rate make this plant a perfect candidate for developing an in vitro regeneration method. However, to date, no such work has been procured in this plant. An efficient method for plant regeneration via shoot organogenesis from callus cultures has been developed using nodal explants in H. isora. Murashige and Skoog (MS) media counting 2,4-Dichlorophenoxyacetic acid (2,4-D, 2.26 to 13.57 μM), Indole-3-acetic acid (IAA, 2.85 to 17.13 μM), Indole-3-butyric acid (IBA, 2.46 to 14.70 μM), 6-Benzylaminopurine (BA, 2.22 to 13.32 μM) and Kinetin (Kin, 2.32 to 13.92 μM) either singly or in the following combinations (IAA + BA; IAA + Kin, and BA + Kin) produced granular callus except BA + Kin which resulted in compact, hard, greenish-white (CHGW) callus. The optimum CHGW callus (2.62 g fresh weight/ explant) was produced on MS media with 13.32 μM BA + 2.32 μM Kin with over 93% callus induction frequency. Optimum shoot organogenesis (67% frequency) was achieved in CHGW callus with lower level of BA (2.22 μM) and Kin (2.32 μM) and produced 3.2 shoots/0.5 g callus within 35 d of culture. Microshoots were rooted successfully (62% frequency) after 35 d of culture on 1/2MS containing 4.90 μM IBA and hardened off. Antioxidant enzymes such as catalase, peroxidase, polyphenol oxidase, and biochemical parameters viz. hydrogen peroxide, reducing and nonreducing sugars, starch, proteins, phenols, and proline contents were studied in regenerating and nonregenerating CHGW calluses to establish a correlation between these parameters and shoot morphogenesis. All the enzyme activities and biochemical parameters were found more in regenerating callus than in nonregenerating except phenols.