The GA application on grapevines induces parthenocarpy, fruit set without fertilization, and the inhibition of pollen tube growth. But the molecular mechanism underlying this inhibition is not understood. Similar defective pollen tube growth within the transmitting tract has been reported in the mutant of GABA transaminase (GABA-T), referred to as pollen-pistil-interaction2 (pop2) in Arabidopsis. In spite of the similarity of pollen tube growth inhibition observed in GA-applied grapevines with that of pop2, only the effects of GABA on stress responses in grapevines have been reported. In present study, transcriptional changes of Vitis GABA metabolic genes, together with changes in GABA levels with or without GA application were analyzed to define how GA application restrained the pollen tube growth in grapevines. A GA solution (Dongbu, Seoul, Korea) at 100 ppm was onto inflorescence clusters 14 days before full bloom (DBF) and clusters were harvested at 0, 1, 2, 4, 7, 9, 12, 14, 16, and 19 days after GA application. Harvested inflorescence samples were immediately frozen in LN2 and extracted RNA and amino acid. The GABA contents were analyzed using high-performance liquid chromatography (Agilent 1100 HPLC, Agilent Technologies, Inc., Santa Clara, USA) equipped with a C18 column (4.6 mm×150 mm, 3.5 μm/VDS optilab, Berlin, Germany), according to the manufacturer’s instructions. Without GA application, the simultaneous high expressions of VvGAD1, VvGAD4 and VvGABA-T2 during 10 to 5 days before full bloom (DBF) showing the activation of GABA metabolism. But the contents of GABA were low before 2 DBF, and it peaked only at near full bloom when expression levels of VvGABA-T2 remained low. After GA application, the contents of GABA were constant during 10 to 5 DBF, although transcription levels of both VvGAD1 and VvGABA-T2 rapidly declined less than 30% of the levels observed without GA application. However, the GABA levels increased more than 2-fold only at near full bloom, compared to those without GA application, and at that time, expression levels of VvGAD1 up-regulated more than 3-fold and those of VvGABA-T2 kept low. But other amino acid contents did not show significant changes. In case of VvSSAHDs, their transcriptional changes with or without GA application were not correlated with GABA levels. These results indicates that GABA levels before pollination is tightly regulated, but GA application alters the GABA-shunt to accumulate excess GABA more than needed for proper pollen tube growth at full bloom. Gibberellin application alters the GABA-shunt to accumulate excess GABA resulting in inhibition pollen tube growth in grapevines.