The objective of this study was to investigate the effect of exogenous nitric oxide on cellular response and production of secondary metabolites in Catharanthus roseus callus. The cell suspension and callus of C. roseus were treated with sodium nitroprusside, and then cell viability, morphology, the amount of H2O2, proline, lipid peroxidation, the activity of superoxide dismutase, catalase and peroxidase were evaluated. Also production of total antioxidant, alkaloid, flavonoid and phenolic compounds were estimated. A Significant (p<0.05) reduction was observed in viability according to the dose of treatment. Also, the amounts fo H2O2, proline, total antioxidant, alkaloid, flavonoid, total phenolic compounds and the activity of the investigated enzymes increased significantly. Malondialdehyde level as a lipid peroxidation indicator was increased significantly in response to treatment. In conclusion, we may announce that the sodium nitroprusside caused cellular membrane damage due to oxidative stress, but an increase in the production of secondary metabolites was observed too. Exogenous NO treatment might be a useful way to increase production of alkaloids at cellular level