Gut microbiota-derived trimethylamine N-oxide (TMAO) has been implicated in both intestinal and renal diseases; however, its specific role in modulating gut-kidney interactions remains unclear. This study aimed to investigate the effects of TMAO on gut-kidney crosstalk using a mouse model of diarrhea. Mice were divided into four groups: normal, model, TMAO, and TMAO + model. The normal group received sterile water, while the other groups were administered adenine + , TMAO, or a combination of TMAO and adenine + . Samples were collected to assess morphological changes in the colon and kidney, evaluate the colonic mucosal barrier and renal function, and measure NLRP3 inflammasome activity and inflammatory cytokine levels in colonic and renal tissues. TMAO levels and the gut microbiota composition were analyzed using 16S rRNA sequencing. The model group exhibited altered stool morphology, which was further aggravated by TMAO intervention. Both the model and TMAO + model groups exhibited significant damage to intestinal and renal tissues, along with compromised intestinal mucosal barriers and impaired renal function compared to controls. Inflammatory markers were elevated in these groups, with the TMAO + model group showing the most pronounced increases. Correlation analysis indicated significant relationships among TMAO levels, inflammasome activation, and inflammatory cytokines. The genera and negatively correlated with TMAO, whereas and genera positively correlated with TMAO. In conclusion, TMAO plays a critical role in modulating gut-kidney crosstalk by promoting inflammation, disrupting mucosal and renal integrity, and altering the gut microbial ecosystem.