Pollen fertility is a crucial factor for successful pollination and essential for seed formation. Recent studies have suggested that a diverse range of internal and external factors, signaling components and their related pathways are likely involved in pollen fertility. Here, we report a single C2-domain containing protein, OsPBP1, initially identified through cDNA microarray analysis. OsPBP1 is a single copy gene and preferentially expressed in pistil and pollen but down-regulated by pollination. OsPBP1 had a calcium concentration-dependent phospholipid-binding activity and was localized mainly in cytoplasm and nucleus, but translocated onto the plasma membrane in response to an intracellular Ca(2+) increase. Pollen grains of antisense OsPBP1 transgenic lines were largely nonviable, germinated poorly in vitro and of low fertility. OsPBP1 protein was localized in a region peripheral to pollen wall and vesicles of elongating pollen tube, and its repressed expression reduced substantially this association and led to alteration of microfilament polymerization during pollen germination. Taken together, these results indicate that OsPBP1 is a novel functional C2-domain phospholipids-binding protein that is required for pollen fertility likely by regulating Ca(2+) and phospholipid signaling pathways.