Insect-transmitted viruses constitute a major threat to rice productivity and thus worldwide food security. To tackle this problem, we have been using Rice stripe virus (RSV), which is transmitted by small brown planthopper (Laodelphax striatellus), as a model to unravel antiviral defense mechanisms in rice that can be harnessed to combat rice virus epidemics. Our previous work revealed a complex interplay between RSV infection and the RNA silencing pathways in rice that determine infection outcomes. We established 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. These and other reports suggest that many miRNAs act as pivotal modulators of plant antiviral immune responses by regulating the turnover of immunity-associated targets. In particular, the expression of miR528 was dramatically down-regulated. However, it is unknown whether the down-regulation is directly caused by lower transcription. Here we show that this miR528-AO defense module is 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. In conclusion, SPL9-mediated transcriptional activation of miR528 expression adds a novel layer of regulation of the miR528-AO antiviral defense.