The present study evaluated the role of nitric oxide (NO) in mediating adventitious root (AR) growth, lignification and related enzymatic changes in the hypocotyls of Vigna radiata. To meet the objectives, the changes in AR growth, lignin content, and the activities of enzymes-peroxidases, polyphenol oxidases, and phenylalanine ammonia lyases- with NO donor and its scavenger were monitored. Hypocotyls were cultivated in aqueous solution supplemented with different concentrations of SNP (sodium nitroprusside, NO donor compound) and its scavenging compound (2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; cPTIO). Specifically, at low concentrations, SNP induced AR growth, increased the total lignin content and altered the activities of related oxidoreductases- peroxidases, polyphenol oxidases and phenylalanine ammonia lyases- which are involved in lignin biosynthesis pathway. At higher concentrations, a decline in AR growth and lignification was noticed. We analysed the function of NO in AR formation by depleting the endogenous NO using scavenging compound cPTIO. Hypocotyls grown in a medium supplemented with scavenger cPTIO exhibited significant decline in AR growth and the activities of lignin synthesizing enzymes. Application of NO scavenger showed that stimulatory properties on root lignification may be owing to NO itself. In addition, changes in AR growth were significantly correlated with these modified biochemical activities. Our analysis revealed that NO supplementation induces prominent alterations in lignin level during AR formation and this might be due to an alteration in the activity of lignin biosynthetic enzymes, which further affected the polymerization of monolignols and AR growth.