Pathogen infection poses a serious threat to the survival and commercial quality of cultured Paa spinosa, which provide protection as a substitute for wild P. spinosa. The gut microbiota plays vital roles in host health and immunity. To provide guidance for preventing intestinal diseases of artificial P. spinosa culture, we compared gut microbiota compositions of wild and cultured P. spinosa using high‐throughput sequencing. A total of 11,526 operational taxonomic units (OTUs) were identified from 14,043 sequences from each sample. Cetobacterium, PW3 Bacteroides and some unidentified species from Bacteroidales, Rikenellaceae and Clostridiales were significantly increased in the gut microbiota from cultured P. spinosa, whereas Faecalibacterium and unidentified bacteria from Ruminococcaceae were significantly decreased in the gut microbiota from cultured P. spinosa. According to the gut microbiota composition, we hypothesized that the cultured P. spinosa in Jing'an would exhibit a higher risk of pathogenic infection than those in Cili. These results provided a method to forecast the pathogenic infection risk of cultured P. spinosa, which could guide the artificial culture of and prevent diseases in P. spinosa through gut microbiota.