Melatonin (MT) and nitric oxide (NO) are known as scavengers of free radicals and an antioxidant against biotic and abiotic stresses in plant defense systems. However, whether NO interplays role in MT-induced antioxidant defense remains to be determined in the plants exposed to lead (Pb) toxicity. So, two experiments were designed to evaluate the role of NO in MT-mediated tolerance of maize plants to Pb stress. In the initial experiment, prior to starting different treatments, a solution of 0.05- or 0.10-mM MT was sprayed every other day for a period of 10 days to the leaves of maize plants exposed to Pb stress (0.1-mM PbCl2). Pb toxicity significantly caused reduction in plant biomass (both fresh and dry), PSII maximum efficiency (Fv/Fm), total chlorophyll, leaf potassium (K), calcium (Ca), and leaf water potential, but it resulted in increased levels of proline, hydrogen peroxide (H2O2), malondialdehyde (MDA), electron leakage (EL), leaf Pb, and endogenous NO. An addition experiment was set up to further understand whether NO played role in mitigation of Pb toxicity in maize plants by MT using scavengers of NO and cPTIO combined with the MT treatments. MT-induced tolerance to Pb toxicity was totally eliminated by cPTIO by reversing endogenous NO. The present results clearly indicated that MT mediated the endogenous NO to improve tolerance of maize plants to Pb toxicity. This evidence was also supported by the increases of H2O2 and MDA and reduces some antioxidant enzyme activities tested as well as the plant growth inhibition and increased leaf Pb content by application of MT combined with cPTIO.