Hexokinase (HXK) has been implicated to be a key player in sugar signaling and metabolism. However, little is known how HXKs regulate growth and development in planta. In rice, there are 10 members of the HXK gene family. One of them, OsHXK10, is preferentially expressed in stamen. This study aims to determine the in vivo role of OsHXK10. The promoter of OsHXK10 was found to drive the expression of β-glucuronidase (GUS) specifically in the anther wall and mature and germinating pollens as well as in palea of rice flower. Interestingly, similar promoter expression pattern was also found in Arabidopsis. Analyses of plants transformed with OsHXK10–green fluorescent protein (GFP) fusion construct suggest that OsHXK10 is localized in the cytoplasm. Suppression of OsHXK10 expression in rice by using RNAi approach showed that the anthers of some flowers in the RNAi lines were unable to dehisce, probably due to inhibition in cell wall thickening of the anthers. The non-dehiscent anther flower (ndaF) failed to shed pollens. Real-time RT-PCR and in situ hybridization analysis revealed that OsHXK10 mRNA level was significantly reduced in the ndaF, particularly in their anther walls, as compared to that of the normal flower. Pollens collected from the RNAi lines appeared to be morphologically normal with similar level of cell wall polysaccharides and starch deposition, as compared with that of the wild-type. However, their germination capacity was markedly decreased. By seed maturity, the proportion of empty seed was significantly increased in the OsHXK10 RNAi lines than that of the wild-type. The results demonstrate that OsHXK10 plays an essential role in anther dehiscence, pollen germination and hence grain filling in rice.