The nanoencapsulation of nitric oxide (NO) donors is an attractive technique to protect these molecules from rapid degradation, expanding, and enabling their use in agriculture. Here, we evaluated the effect of the soil application of chitosan nanoparticles containing S-nitroso-MSA (a S-nitrosothiol) on the protection of soybeans (Glycine max cv. BRS 257) against copper (Cu) stress. Soybeans were grown in a greenhouse in soil supplemented with 164 and 244 mg kg−1 Cu and treated with a free or nanoencapsulated NO donor at 1 mM, as well as with nanoparticles without NO. There were also soybean plants treated with distilled water and maintained in soil without Cu addition (control), and with Cu addition (water). The exogenous application of the nanoencapsulated and free S-nitroso-MSA improved the growth and promoted the maintenance of the photosynthetic activity in Cu-stressed plants. However, only the nanoencapsulated S-nitroso-MSA increased the bioavailability of NO in the roots, providing a more significant induction of the antioxidant activity, the attenuation of oxidative damage, and a greater capacity to mitigate the root nutritional imbalance triggered by Cu stress. The results suggest that the nanoencapsulation of the NO donors enables a more efficient delivery of NO for the protection of soybean plants under Cu stress.