The objective of this study was to understand the effect of long-term aconitine (AC) oral administration on the digestive tract and serum metabolism. Subjects consumed either 0.9% NaCl (n = 8) or AC (n = 17) gavage designed to represent human chronic AC administrations for 13 days. Organ pathology was determined using hematoxylin-eosin staining and immunohistochemistry. Fecal and proximal intestinal content samples were collected to perform shotgun metagenomic sequencing. Serum samples were collected, and untargeted metabolomics was performed. In this study, AC administration induced proximal intestine, liver, and kidney injury. Microbiome composition remained stable after AC exposure, while several microbes presented dynamic alteration. Moreover, AC affected the abundance of the fatty acid biosynthesis rate-limiting gene accA at day 7. AC induces 30 serum metabolites to significantly change at day 14, including several short-chain acylcarnitines. WGCNA revealed 2 sub-modules associated with the level of several short-chain acylcarnitines. In summary, AC affects the digestive tract and serum metabolism after chronic administration. AC may affect the enrichment of microbial-derived accA gene. The abundance of serum acylcarnitines detected in the AC group may associate with its anti-heart failure effects.