The pollen grains of most angiosperms contain stores of RNAs and their translation products required for pollen germination and subsequent early elongation of pollen tubes. Polypyrimidine tract-binding protein (PTB), which is involved in the regulation of pre-mRNA alternative splicing, internal ribosomal entry site (IRES)-mediated translation and mRNA localization/sorting, is known to act as a bridging molecule between RNAs and a variety of cellular factors to fulfill cellular functions in both the nucleus and cytoplasm. Moreover, it has been reported that PTB plays roles in the differentiation and development of animal cells and tissues. In the Arabidopsis genome, there are two PTB-related genes, tentatively termed AtPTB1 and AtPTB2. In the present study, the physiological functions of AtPTBs were investigated using genetic and cytological approaches. The AtPTB promoter was highly active in vegetative cells of mature pollen grains, and AtPTB was localized in the nucleus and cytoplasm of these vegetative cells. Mutations in the AtPTB genes resulted in decreased germination efficiency, and this effect was rescued by introduction of the AtPTB2 promoter::AtPTB2-GFP. Taken together, these findings suggest that AtPTB is involved in pollen germination through possible RNA metabolism processes in late-maturing and mature pollen grains.