Maize is not only a staple food crop but also an important raw material for feed and industry; however, the threat of weeds leads to a serious decline in its output and quality. The G2-aroA gene confers glyphosate herbicide tolerance to crops. In this study, the food safety of genetically modified (GM), glyphosate-tolerant maize with the G2-aroA gene was evaluated in a 90-day feeding study in Sprague-Dawley (SD) rats. Maize grain from GM or non-GM isogenic control lines were separately formulated into rodent diets at concentrations of 12.5% (low level), 25% (middle level), and 50% (high level). An additional group of rats were fed a commercialized diet as a control. The toxicological response variables, including body weights, food consumption, serum biochemistry, hematology, and absolute and relative organ weights, were compared between rats fed GM maize and those fed non-GM maize after consumption of test diets for 90 days. In addition, gross and microscopic pathology were conducted among treatment groups. No adverse effects related to the consumption of GM maize were detected in the subchronic feeding study. These results indicated that the GM glyphosate-tolerant maize was as safe and nutritious as conventional maize.