Abstract: Paa spinosa is an important amphibious economic animal distributed in the rolling mountains in the south of China. In the process of artificial farming, diseases have become an important factor on its yield reduction. Intestinal microbiota is considered to be involved in organism’s immune response. However, it is unclear whether the composition and diversity of intestinal microbiota are related to the health condition of P. spinosa. To explore the structure of intestinal flora of P. spinosa under different health conditions, 13 healthy (Health), 19 wonky-head disease (WHD) and 18 blue-eye disease (BED) P. spinosa were sampled. Total intestinal DNA were extracted following manufacture’s procedures, and V4-V5 region of 16S rRNA were amplified and sequenced using Illumina Miseq sequencing platform. A total of 3984 operational taxonomic units (OTUs) were obtained, belonging to 35 phyla, 80 classes, 155 orders, 283 families, and 501 genera. The results showed that the microbiota abundance of the healthy group was significantly higher than that of the sick groups with no significant difference between group WHD and BED (Chao1 index: Health: 1232.92; WHD: 975.57; BED: 1048.76), and the microbial community diversity exhibited no significant difference between healthy and sick groups (Shannon index: 5.27; 5.20; 5.41). The dominant bacterial phyla included Firmicutes (52.08%; 49.57%; 26.48%), Bacteroidetes (30.55%; 21.93%; 43.02%) and Proteobacteria (15.55%; 10.42%; 5.25%). As a consequence, Deferribacteres increased significantly in sick groups (0.18%; 14.05%; 22.16%). The dominant bacterial genera included Bacteroides (29.70%; 16.09%; 21.76%), an unclassified Erysipelotrichaceae (21.38%; 6.92%; 4.94%), an unclassified Lachnospiraceae (12.23%; 15.98%; 4.43%), Citrobacter (10.31%; 6.19%; 1.10%) and Eubacterium (9.09%; 4.88%; 0.54%). Mucispirillum (0.17; 13.89%; 21.94%) of Deferribacteres increased significantly in sick groups. The analysis of Beta diversity showed that samples from healthy and sick groups were clustered separately. These results reveal that the structure and diversity of intestinal microbiota were related to the health conditions of P. spinosa. This study provides reliable data and theoretical guidance for the subsequent improvement of the production and quality of P. spinosa, and the selection of intestinal probiotics and the study of probiotic preparations.