Soybean is an important food resource for the eastern countries and herbicide-tolerant genetically modified soybeans (GMS) were widely developed to deal with weeds problems. Unprocessed soybean flour instead of dehulled and defatted soybean meal was used to reflect the safety of soybean food in whole. Rats were given formulated diets containing DP-356ϕ43 or non-GM soybean JACK at an incorporation rate of 7.5%, 15%, or 30% (w/w), respectively for 90 days. Targeted traditional toxicological response variables were measured to reflect the holistic health of animals. No treatment-related adverse or toxic effects were observed based on an examination of the daily clinical signs, body weight, food consumption, hematology, serum biochemistry, and organ weight or based on gross and histopathological examination. The results demonstrate that the soybean DP-356ϕ43 is as safe for consumption as conventional soybean JACK. In the current study, the effect of a herbicide-tolerant GMS DP-356043 on identified intestinal microbiota was evaluated in a rodent feeding study compared with its conventional control JACK. Feces samples from rats consuming different diets were collected before the start of the experiment (time 0) and at monthly intervals (at the end of the 1st, 2nd and 3rd months) over the course of 90 days. Six types of bacterias shared by humans and rats were detected with Q-PCR. The results of QPCR indicated that the GMS 356ϕ43 had a comparable effect on the abundance of Bifidobacterium group, Clostridium perfringens subgroup, Escherichia coli, and Bacteroides–Prevotella group as the non-GMS JACK.