Nitric oxide (NO) is as a signaling molecule that participates in the regulation of plant development and in a number of physiological processes. However, the function and regulatory pathway of NO in the growth and development of edible mushrooms are still unknown. This study found that NO played a negative role in the transformation of from vegetative growth to reproductive growth by the exogenous addition of NO donors and scavengers. Further studies showed that NO can inhibit the gene expression and enzyme activity of aconitase (ACO). Moreover, the overexpression (OE) of mitochondrial and RNA interference (RNAi) confirmed that ACO participates in the regulation of the primordia formation rate. The effects of OE and RNAi on the tricarboxylic acid (TCA) cycle and energy metabolism were further measured. The results showed that RNAi- mutant strains can affect the enzyme activities of isocitrate dehydrogenase of mitochondria (ICDHm) and α-ketoglutarate dehydrogenase (α-KGDH) in the TCA cycle, thereby reducing the production of nicotinamide adenine dinucleotide (NADH) in the TCA cycle, decreasing the contents of adenosine triphosphate (ATP) and hydrogen peroxide (HO), and negatively regulating the rapid formation of primordia. In addition, HO was significantly increased during the transformation from vegetative growth to reproductive growth of . Additionally, the exogenous addition of HO and its scavengers further confirmed the positive regulation by HO in primordia formation. This study shows that during the growth and development of , NO can inhibit the expression of the mitochondrial gene and ACO protein in the TCA cycle, reduce the production of ATP and HO in the respiratory chain, and negatively regulate the rate of primordia formation.