Nitrite (NIT) is a toxic nitrogenous compound that adversely affects living organisms. This study utilized pathological, metagenomic, and transcriptomic analyses to examine the toxic effects and damage mechanisms of NIT exposure on the intestine of Procambarus clarkii. Findings revealed that excessive NIT led to pathological changes in the crayfish intestine, an imbalance in intestinal flora, and an increase in harmful bacteria such as Enterobacteriaceae, Shewanella, and Vibrionaceae. Additionally, NIT exposure caused various physiological changes, including oxidative stress, immune imbalance, metabolic disorders, excessive autophagy, and apoptosis, leading to inflammatory damage in the intestine. Moreover, NIT exposure disrupted crayfish energy metabolism by reducing ATP content and disturbing energy homeostasis. The inhibition of the intestinal immune system is speculated to disrupt intestinal flora homeostasis and promote the overgrowth of opportunistic pathogenic bacteria, increasing the risk of crayfish infections. This study provides new insights into the antioxidant and immune defense mechanisms against NIT in crayfish and may aid in developing strategies to enhance disease and stress resistance in crustaceans.