In contrast to animals, in which products of meiosis differentiate directly into sperm, flowering plants employ a specific mechanism to give rise to functional sperm cells, the specifics of which remain largely unknown. A previous study revealed that, compared to yeast and vertebrates, which have two proteins (Rad21 and its meiosis-specific variant Rec8) that play a vital role in sister chromatid cohesion and segregation for mitosis and meiosis, respectively, the rice genome encodes four Rad21/Rec8 proteins (OsRad21s). In this paper, phylogenetic and immunostaining analyses reveal that OsRad21-3 is an orthologue of yeast Rad21. OsRAD21-3 transcript and protein accumulated preferentially in flowers, with low levels in vegetative tissues. In flowers, they persisted from the stamen and carpel primordia stages until the mature pollen stage. OsRAD21-3-deficient RNAi lines showed arrested pollen mitosis, aberrant pollen chromosome segregation and aborted pollen grains, which led to disrupted pollen viability. However, male meiosis in these RNAi lines did not appear to be severely disrupted, which suggests that the main involvement of OsRAD21-3 is in post-meiotic pollen development by affecting pollen mitosis. Furthermore, of the four OsRAD21 genes in the rice genome, only OsRAD21-3 was expressed in pollen grains. Given that the mechanism involving generation of sperm cells differs between flowering plants and metozoans, this study shows, in part, why flowering plants of rice and Arabidopsis have four Rad21/Rec8 proteins, as compared with two in yeast and metozoans, and gives some clues to the functional differentiation of Rad21/Rec8 proteins during evolution.