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Objective To investigate the composition and distribution characteristics of intestinal flora in patients with epilepsy at middle-high altitude, in order to provide theoretical reference for the etiological study of epilepsy. Methods From September 2021 to August 2022, we continuously recruited middle-high altitude epilepsy (MHE) patients (altitude 2200-4500 m) and healthy people at the same altitude as middle- high altitude normal (MHN). MHE were epileptic patients in neurology department of Qinghai Provincial People's Hospital, and MHN were healthy people in physical examination center of Qinghai Provincial People's Hospital which matched 1∶1 with MHE by age and gender. Stool tissue specimens from both groups were collected, and the 16S rDNA V3 to V4 regions of the intestinal flora were sequenced for DNA and analyzed bioinformatically. Results A total of 32 cases of MHE and 33 cases of MHN who met the inclusion and exclusion criteria. Alfa diversity analysis showed that ACE index, Shannon index and Chao index in MHE group were lower than those in MHN group (all P < 0.05). Beta diversity analysis showed that there were significant differences in the distribution of intestinal flora between the two groups. In the analysis of intestinal flora composition, MHE group also showed different characteristics from MHN group at phylum level and genus level. The relative abundance of Verrucomicrobiota, Collinsella, Fusobacteriota, Bifidobacterium and other bacteria increased, whereas the relative abundance of Bacteroidota, Bacteroides, Prevotella, Faecalibacterium and other bacteria decreased. Conclusion The intestinal flora of epilepsy patients at middle and high altitude has different polymorphisms from that of healthy people at the same altitude, which may be associated with epilepsy.