Glyphosate-resistant (GR) transgenic soybean has never been cultivated commercially in China. It is essential to develop the separation measures required to prevent out-crossing between GR and conventional soybean (Glycine max (L.) Merr.) by characterizing the transgene flow before GR soybean is released. In this study, the transgene flow from a GR transgenic soybean AG5601 to conventional soybeans was characterized. First, natural out-crossing rate was evaluated using 36 conventional soybean varieties interplanted with GR soybean AG5601 transformed with a cp4 EPSPS gene conferring the resistance to herbicide glyphosate in the field in 2007 and 2008 in China. Second, drift distance of cp4 EPSPS gene from GR soybean AG5601 to soybean cv. Zhonghuang13 was evaluated using the progenies harvested from eight directions at different distance. Third, the relationship of gene flow of GR soybean AG5601 with flowering synchronization days or insect pollinators of each variety was analyzed using regression analysis. Thirty-two of 36 tested conventional soybean varieties had surviving progenies after two times of sprays of glyphosate, and 49 of 41,679 progenies were verified to be glyphosate-tolerant heterozygous offspring. The out-crossing rates in positive varieties (having surviving offspring after two times of sprays of glyphosate) ranged from 0.039 to 0.934 %. The farthest distance (drift distance) between soybean AG5601 and cv. Zhonghuang13 at which out-pollinating was still able to be observed was 15 m, with an out-crossing rate of 0.012 %. Regression analysis showed that there was a positive relationship between cross-pollination frequency and flowering synchronization days or pollinator insects. Therefore, when GR soybean is released to the field, it should be critically separated with the conventional soybean in space and cultivation time with efficient insect control during flowering.