Panax notoginseng is a traditional medicinal herb in China. However, the high capacity of its roots to accumulate cadmium (Cd) poses a potential risk to human health. Our previous study showed that nitrate reductase (NR)-dependent nitric oxide (NO) production promoted Cd accumulation in P. notoginseng root cell walls. In this study, the role of Mg in the regulation of NO production and Cd accumulation in P. notoginseng roots was characterized. Exposure of P. notoginseng roots to increasing concentrations of Cd resulted in a linear increase in NO production. The application of 2 mM Mg for 24 h significantly alleviated Cd-induced NO production and Cd accumulation in roots, which coincided with a significant decrease in the NR activity. Western analysis suggested that Mg increased the interaction between the 14-3-3 protein and NR, which might have been a reason for the Mg-mediated decrease in NR activity and NO production under Cd stress. These results suggested that Mg-mediated alleviation of Cd-induced NO production and Cd accumulation is achieved by enhancement of the interaction between the 14-3-3 protein and NR in P. notoginseng roots.