Nano-titanium dioxide (nTiO₂) has been reported to improve tolerance of plants against different environmental stresses by modulating various physiological and biochemical processes. Nitric oxide (NO) has been shown to act as an important stress signaling molecule during plant responses to abiotic stresses. The present work was planned to investigate the involvement of endogenous NO in nTiO₂-induced activation of defense system of fava bean (Vicia faba L.) plants under water-deficit stress (WDS) conditions. Water-suffered plants showed increased concentration of hydrogen peroxide (H₂O₂) and superoxide (O₂^-) content coupled with increased electrolyte leakage and lipid peroxidation which adversely affected nitrate reductase (NR) activity, chlorophyll content and growth of the plants. However, application of 15 mg L^-1 nTiO₂ to stressed plants significantly induced NR activity and synthesis of NO which elevated enzymatic and non-enzymatic defense system of the stressed plants and suppressed the generation of H₂O₂ and O₂^- content, leakage of electrolytes, and lipid peroxidation. Application of nTiO₂, in association with NO, also enhanced the accumulation of osmolytes (proline and glycine betaine) that assisted the stressed plants in osmotic adjustment as witnessed by improved hydration level of the plants. Involvement of NO in nTiO₂-induced activation of defense system was confirmed with NO scavenger cPTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] which caused recurrence of WDS.