Plant Xrn4 is a cytoplasmic 5' to 3' exoribonuclease that is reported to play an antiviral role during viral infection as demonstrated by experiments using the Xrn4s of Nicotiana benthamiana and Arabidopsis thaliana. Meanwhile, little is known about the anti-viral activity of Xrn4 from other plants. Here, we cloned the cytoplasmic Xrn4 gene of Oryza sativa (OsXrn4), and demonstrated that its over-expression elevated the 5'-3' exoribonuclease activity in rice plants and conferred resistance to rice stripe virus, a negative-sense RNA virus causing serious losses in East Asia. The accumulation of viral RNAs was also decreased. Moreover, the ectopic expression of OsXrn4 in N. benthamiana also conferred plant resistance to tobacco mosaic virus infection. These results show that the monocotyledonous plant cytoplasmic Xrn4 also has an antiviral role and thus provides a strategy for producing transgenic plants resistant to viral infection.