Although it has been reported that γ-amino butyric acid (GABA) plays an important role in pollen tube development, the molecular mechanisms of this function, especially, its role in pistil-pollen tube interaction, are largely unknown.Here, we present evidences to show how GABA signaling works in regulating pollen tube growth.Our results showed that there is a GABA gradient from the stigma to the ovary in tobacco pistils.Exogenous GABA modulates Ca2+-permeable channels on the pollen and pollen tube plasma membrane and mediates the communication between the pistil and the pollen tube.Whole-cell voltage-clamp experiments and non-invasive micro-measurement techniques (NMT) confirmed Ca2+ vibration in pollen tubes in responding to GABA signals.We also demonstrated that the rate-limiting enzyme of GABA biosynthesis, glutamate decarboxylase (GAD) expression/function is affected by Ca2+/calmodulin (CaM), participates in the calcium signaling network, and is required for the maintenance of microfilament organization and vesicle directional trafficking.Quantitative RT-PCR assays indicated that at least three downstream signaling pathways, PLC3, PLD, and CDPK2, might integrate the exogenous and endogenous GABA signals to regulate actin dynamics and directional vesicle trafficking, and eventually regulate pollen tube tip growth.Our findings revealed that GABA in pistil regulates pollen tube growth via activating Ca2+-permeable channels and modulating downstream Ca2+/CaM-GAD signaling networks.