Heat stress is a potent modulator of the avian neuroendocrine system with concomitant impact on the gut microbiome. As an interkingdom signaling molecule, serotonin is largely derived from the gut and found in large concentrations in the avian gut lumen. Despite the role of serotonin in animal stress physiology and related host-microbe interactions, whether heat stress alters avian enteric concentrations of serotonin is unknown. As such, the present study sought to determine whether acute or chronic exposure to moderate heat stress alters both enteric serotonin concentrations and the microbiome in the chicken gut. Chickens were, or were not, subjected to an acute (1 day), repeated acute (2 days) or chronic (6 days) moderate ambient cyclic heat stress (12h per day, 31°C). Enteric concentrations of serotonin were significantly decreased in the acute heat stress group (P <
0.05), and rebounded to become elevated in the chronic heat stress group (P <
0.05). Shotgun metagenomic sequencing revealed heat stress caused both functional and taxonomic changes in the cecal microbiome. Abundances of bacterial taxa that are known to interact with the host via the serotonergic system, including Lactobacillus spp., and Bifidobacterium spp., were significantly (P <
0.05) altered by heat stress. As these findings demonstrate that heat stress can alter serotonin concentrations in the chicken intestinal tract, with distinct outcomes depending on duration of the stressor, serotonergic signaling may serve as potential leverageable point of intervention in host-microbe interactions including foodborne pathogen colonization in the chicken gut. In addition, this study provides novel insight into the impact of acute and chronic heat stress on the avian microbiome, and its relationship to stress-driven changes in the enteric serotonergic system.