In order to accumulate γ-aminobutyric acid (GABA), soybean seeds (cultivar Jindou 25) were germinated for 102 h at different temperatures (19, 25 and 32 °C). The content of GABA, glutamic acid and the activity of the glutamate decarboxylase (GAD) and GABA transaminase (GABA-T) in soybeans during germination were investigated. The results showed that the germination temperature and germination time had great influences on GABA content and the related enzyme activities in soybean seeds. As compared to raw soybeans, an increase in the content of GABA and glutamic acid was observed, as well as GAD activity in soybeans during germination, while germination at 32 °C was better for accumulating GABA in soybeans. The GABA-T activity first decreased and then increased at 19 °C and 25 °C, on the contrary, it first increased and then decreased sharply during germination at 32 °C. These results indicate that the increase of GABA content can be attributed to the changes of GAD and GABA-T activities rather than enough glutamic acid resulting from the degradation of protein during germination of Jindou 25 seeds. However, more assays need to be further performed with more soybean cultivars