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Canola is one of the most important plant oilseed crops. To avoid the threat of herbicides, the RF3 line with bar gene and barstar gene was developed, which can act as glufosinate resistance resources and restore fertility in hybrid lines. To assess the food safety of transgenic canola RF3, 2.5%, 5% and 10% GM canola RF3 and its non-GM isogenic line Drakkar were formulated into diet to feed Spragure-Dawley (SD) rats for 90 days. The effects on the general growth and toxicological parameters, as well as gut microbiota of rats, were evaluated. Several significant differences on body weight, feed consumption, relative organ weight, hematology and serum biochemistry were observed among rats in the 90-day feeding test. However, these statistical differences were randomly observed among different groups and were not dose-related, which were not considered to be biologically significant. Furthermore, the results of bacterial 16S rRNA sequencing of fecal samples showed that the diets containing GM canola did not disturb the balance of gut microbiota. In conclusion, the canola RF3 is considered as safe and wholesome as the non-GM canola based on this 90-day feeding test and gut microbiota analysis.Several differences found in 90 d feeding test were not related to the GM canola RF3.GM canola didn't disturb the balance of gut microbiota based on 16S rRNA sequencing.The canola RF3 is considered as safe and wholesome as the non-GM canola based on this test.