Climate warming and frequent incidents of extreme high temperatures are serious global concerns. Heat stress induced by high temperature has many adverse effects on animal physiology, especially in aquatic poikilotherms. Chinese mitten crab (Eriocheir sinensis) is sensitive to high temperatures, this study evaluated the harmful effects of heat stress on the neurotoxicity, intestinal health, microbial diversity, and metabolite profiles. The results showed that heat stress caused histopathological damages and altered the ultrastructure of lesions in the cranial ganglia. Heat stress significantly upregulated the mRNA expression of apoptosis-related genes, and significantly altered the expression of neurotransmitter receptors. In addition, heat stress induced significant intestinal damages that mainly manifested as a significant increase in the activity of diamine oxidase in the serum and contents of histamine in the intestine. The diversity and abundance of intestinal microbiota altered abnormally in E. sinensis exposed to heat stress, and the bacteria that exhibited significant variations in abundance were closely related to the production of neurotransmitters and neuromodulators. Heat stress caused significant changes in the intestinal metabolite profiles, which mainly involved the amino acid and lipid metabolism pathways. Analysis of the correlation showed that the abnormal changes in metabolites were closely related to differences in the abundance of intestinal microbiota. Therefore, this study showed that heat stress could cause neurophysiological toxic effects, which may be related to intestinal ecological imbalance.