1. The avian gut hosts a complex and dynamic microbial ecosystem, which is essential for regulating host organ function. However, the relationship between the gut microbiota and the hypothalamic axis in acute stress vulnerability in ducks remains unclear.2. This study investigated how the gut microbiota affects microbial metabolism and the host stress response by comparing hypothalamic neurotransmitter availability, microbial composition and co-metabolites generated by both the microbiota and hypothalamus in ducks exhibiting the lowest active avoidance (LAA) and highest active avoidance (HAA) behaviour.3. The HAA group experienced a significant increase in the availability of arginine, histidine, glutamine, norepinephrine, L-tyrosine and melatonin during acute stress in the hypothalamus, compared to that in the LAA group. The 16S rRNA sequencing revealed significant differences in the gut microbiota composition based on acute stress vulnerabilities.4. Both caecal and hypothalamic metabolomic analyses identified 71 metabolites altered in caecal content and 95 in the hypothalamus. There was significant enrichment in pathways such as the cGMP-PKG signalling, dopaminergic synapse and endocrine resistance.5. Correlation analyses demonstrated that certain co-metabolites, including 1,3-dicyclohexylurea, 1-deoxyvaleric acid, 2-amino-2-methyl-1,3-propanediol, 3-chloroaniline, methenamine, N4-acetylcytidine-triphosphate and traumatin, may play a role in the gut microbiota-hypothalamic axis.6. The results suggested that the gut microbiome influenced acute stress responses. This provided a basis for understanding gut-hypothalamic communication and its impact on behaviour in ducks.