Abstract This study aimed to identify whether microbial interactions in the rectum contribute to Shiga toxin producing bacteria colonization. In total, 12 rectal digesta samples based on the previously identified Shiga toxin 2 gene (stx2) abundance (DNA) and expression (RNA) in Shiga toxin-producing bacteria (Stx2- group: detectable DNA, n=6; Stx2+ group: detectable DNA and RNA, n = 6) were subjected to microbial profiling using amplicon sequencing. Firmicutes (72.7 ± 2.0 %) and Bacteroidetes (24.6 ± 1.9 %) are the most predominant phyla of rectal microbiota, and no compositional differences were identified between two groups at the phylum level. The Shannon and Chao1 indices weren’t different in rectal digesta microbial communities between two groups. Twenty-four and thirteen taxa were identified to be group-specific genera in microbial communities from Stx2- and Stx2+ group, respectively (2 out of 6, average relative abundance >0.1%). The network analysis indicated 12 and 14 keystone taxa (Generalists, densely connected with other taxa) in microbial communities between Stx2- and Stx2+ groups, respectively. Eight out of 12 and six out of 14 generalists in the Stx2- and Stx2+ group are belonging to group-specific genera, respectively. Generalists belonging to group-specific genera were broadly distributed in Stx2- network while centralized distributed in the Stx2+ network, suggesting the higher stability of the Stx2- network structure in comparison of Stx2+ network computed by the natural connectivity measurement. However, 66 core genera shared by microbial communities between two groups were not classified into network generalists. Overall, our results indicate microbial crosstalks and keystone taxa in microbial communities between two groups differed, suggesting that the microbial interactions rather than the shifts in taxa abundance may be more important affecting host. Moreover, group-specific genera play a vital ecological role in the microbial interactions, indicating the potential for being microbial markers to differentiate Shiga toxin-producing bacteria colonization in beef cattle.