BACKGROUND: Nutrients are one of the key determinants of gut microbiota variation. However, the intricate associations between the amino acid (AA) profile and the dynamic fluctuations in the gut microbiota and resistome remain incompletely elucidated. Herein, we investigated the temporal dynamics of AA profile and gut microbiota in the colon of pigs over a 24-hour period, and further explored the dynamic interrelationships among AA profile, microbiota, and resistome using metagenomics and metabolomics approaches. RESULTS: JTK_circle analysis revealed that both the AA profile and the gut microbiota exhibited rhythmic fluctuations. With respect to the feed intake, all AAs except L-homoserine (P CONCLUSIONS: Taken together, the AA profile and the gut microbiota exhibit robust fluctuations over a day. The AA profile can affect the gut microbiota and resistome in a direct or indirect manner. These findings may provide new insights into a potential strategy for manipulating the gut microbiota and resistome.