Lead (Pb) is a serious threat for the plant growth and metabolism. Utilization of natural products, such as C-Phycocyanin, to ameliorate heavy metal stress is a successful and ecofriendly strategy for protection and enhancement of tolerance in plants from contaminated conditions. Nanotoxicological effects of lead nanoparticles (Pb NPs) and Pb NPs bound to C-Phycocyanin (C-PC) on vegetative growth, chemical composition, oxidative burst, and DNA integrity in alfalfa plants (Medicago sativa L.) using the plant as a model organism were studied. Pb NPs at different concentrations (100–1000 mg L⁻¹) with and without C-Phycocyanin were examined. Results of the vegetative characteristic results indicated that the Pb NP exposure remarkably reduced plant height, branch number, and dry biomass and leaf area in a dose-response manner. Chemical composition analysis showed that the contents of chlorophyll, nitrogen, phosphorus, potassium and zinc decreased while calcium increased under Pb stress. Exposure to Pb NPs also induced oxidative stress, increasing the levels of catalase, superoxide dismutase, ROS, lipid peroxidation and DNA damage. However, addition of C-Phycocyanin largely ameliorated such alterations thereby stimulating growth and maintaining a good biochemical homeostasis which protected cellular/genetic integrity from damage caused by Pb. It was demonstrated that C-Phycocyanin could be an efficient natural antioxidant and anti-lead nanoparticle protective agent, as well as plant defensive compound to combat stress of heavy metal.