The linkage between Qi and mitochondria was investigated by exploring the effect of Traditional Chinese Medicine (TCM) Qi-invigorating herbs on mitochondrial function at the biochemical and molecular levels. Three Chinese herbs (Astragali radix, Herba cistanche, Panax ginseng) were used to treat cultured mouse kidney cells and the generation of adenosine triphosphate (ATP) was measured. The Qi-invigorating herb, Astragali radix, was selected for further study using additional biological and molecular parameters, including ATP, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mtDNA copies, superoxide dismutase (SOD), glutathione (GSH), cell growth, cell viability and transcriptomes. We also chose two concentrations of Astragali radix to study the hormetic effect. The results indicated that: 1) Qi-invigorating herbs have significant effects on the function of mitochondria, with ATP production and the antioxidant capacity being significantly enhanced, and ROS levels being reduced, allowing for a more optimal oxidation environment. The effect of the herbs followed a hormetic curve with a stimulating effect at lower doses but an inhibiting effect at high doses; 2) The growth of the cells was not affected despite numerous biochemical changes associated with mitochondrial function, indicating the powerful ability of mitochondria to maintain cellular homeostasis; 3) The up-regulation of NOCT gene, related to nicotinamide adenine dinucleotide (NADH) synthesis, offers a molecular basis for the ATP-promoting effect of the Qi-invigorating herbs. This work provides additional insight into the efficacy of TCM herbs from a western perspective.