Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Constipation is a prodromal symptom of PD. It is important to investigate the pathogenesis of constipation symptoms in PD. Rotenone has been successfully used to establish PD animal models. However, the specific mechanism of rotenone-induced constipation symptoms is not well understood. In this work, we found that constipation symptoms appeared earlier than motor impairment in mice gavaged with a low dose of rotenone (30 mg/kg·BW). Rotenone not only caused loss of dopaminergic neurons and accumulation of α-synuclein, but also significantly reduced serum 5-HT levels and 5-HTR4 in the striatum and colon. The mRNA expression of aquaporins, gastrointestinal motility factors (c-Kit, Cx43, smMLCK and MLC-3) in mouse colon was also significantly regulated by rotenone. In addition, both colon and brain showed rotenone-induced inflammation and barrier dysfunction; the PI3K/AKT pathway in the substantia nigra and colon was also significantly inhibited by rotenone. Importantly, the structure, composition and function of the gut microbiota were also significantly altered by rotenone. Some specific taxa were closely associated with motor and constipation symptoms, inflammation, and gut and brain barrier status in PD mice. Akkermansia, Staphylococcus and Lachnospiraceae_UCG-006 may play a role in exacerbating constipation symptoms, whereas Acinetobacter, Lactobacillus, Bifidobacterium, Solibacillus and Eubacterium_xylanophilum_groups may be beneficial in stimulating gastrointestinal peristalsis, maintaining motor function and alleviating inflammation and barrier damage in mice. In conclusion, low-dose rotenone can cause parkinsonism with constipation symptoms in mice by disrupting the intestinal microecosystem and inhibiting the PI3K-AKT pathway and gastrointestinal motility.