Many microRNAs (miRNAs) are critical regulators of plant antiviral defense. However, little is known as to how these miRNAs respond to virus invasion at the transcriptional level. We established previously that defense to Rice stripe virus (RSV) invasion entailed a reduction of miR528 accumulation in rice, thus alleviating miR528-mediated degradation of L-Ascorbate Oxidase (AO) mRNA, bolstering the antiviral activity of AO. Here we show that this miR528-AO defense module is also regulated by the transcription factor SPL9. SPL9 displayed high affinity binding to specific motifs within the promoter region of miR528. Loss-of-function mutations in SPL9 correlated with a significant reduction of miR528 but a substantial increase of AO mRNA, enhancing rice resistance to RSV. Conversely, transgenic overexpression of SPL9 stimulated the expression of miR528, hence lowering the level of AO mRNA and compromising rice defense to RSV. Importantly, gain in RSV susceptibility did not occur when SPL9 overexpression was attempted in mir528 loss-of-function mutants, or in transgenic rice expressing a miR528-resistant AO. In conclusion, SPL9-mediated transcriptional activation of miR528 expression adds a novel layer of regulation of the miR528-AO antiviral defense.