To explore the influence of Bt protein on intestinal bacterial diversity of Pardosa pseudoannulata, we analyzed its intestinal bacteria of Pardosa pseudoannulata feed by fruit fly cultured with cry1Ab protein medium and regular medium using 16S rDNA molecular analysis methods. Study revealed that Pardosa pseudoannulata intestinal bacteria were rich, with intestinal bacterial abundance of female spider higher than male spider. Its dominant bacteria were Brevibacterium, Brevundimonas, Staphylococcus, Lactobacillus, Comamonas, Devosia, Shewanella, Leucobacter, Brachybacterium, Clostridium; the Bt protein had a certain effect on intestinal bacterial diversity of Pardosa pseudoannulata, and the influence of Bt protein to Actinomycetes in intestinal bacteria of Pardosa pseudoannulata was the minimum, to Lactobacillus and Clostridium was the largest.