In order to study pollen-mediated gene flow in transgenic maize (Zea mays L.) in the Huang-huai-hai region of China, field trials were conducted in Jinan, Shandong Province in 2006 and 2007. The frequencies of gene flow from the donor plots, planted with transgenic maize as a pollen source, to the receptor plots, planted with non-transgenic maize, under different temporal or spatial separations were evaluated. The results showed that the frequency of pollen-mediated gene flow of the als gene from transgenic maize to non-transgenic maize decreased significantly with increasing distance. No gene flow was detected at 300 m. At a distance of 30 m, delaying the planting date of the transgenic maize by 1 week decreased the frequency of gene flow by 70%. A delay of 2 weeks decreased the gene flow frequency by more than 90%, while no gene flow was seen when the sowing date was delayed by 3 weeks. The results suggest that an appropriate isolation distance of 300 m or a temporal separation of 3 weeks could prevent gene flow from transgenic maize to non-transgenic maize in the Huang-huai-hai region.