Background and purpose: Several analgosedative drugs used in the management of critically ill patients impair gastrointestinal (GI) propulsion and thereby carry a risk for developing sepsis. The gut microbiota has emerged as a factor that can influence GI motility, but whether GI microbial disruption modifies GI peristalsis impairment by analgosedative drugs has not yet been analysed. This question was addressed in the guinea-pig small intestine following antibiotic-induced depletion of the GI microbiome. Experimental approach: Guinea-pigs were enorally pretreated with meropenem, neomycin and vancomycin, and antibiotic-induced depletion of the GI microbiome was confirmed by 16S rDNA sequencing. Peristalsis in the isolated guinea-pig small intestine was evaluated by assessing the peristaltic pressure threshold at which a peristaltic wave is triggered. The expression of factors that may be relevant to communication between GI microbiota and motor system was examined at the mRNA (qPCR) and/or protein (ELISA) level. Key results: Antibiotic treatment disturbed the small intestinal microbiome as shown by a decrease of bacterial load and α-diversity. Microbial disruption did not affect peristalsis at baseline but blunted the ability of α2-adrenoceptor (ADRA2) agonists to inhibit peristalsis, while the anti-peristaltic effects of sufentanil, midazolam, neostigmine and propofol were inconsistently affected. These functional alterations were complemented by a decreased expression of ADRA2, TLR3, TLR4 and TLR7, IFN-γ and NOS2. Conclusions and implications: Antibiotic-induced disturbance of the GI microbiome selectively blunts the ability of ADRA2 agonists to impair peristalsis. This effect is explained by decreased ADRA2 expression, which may arise from TLR downregulation in the dysbiotic gut.