Despite the fact that a large number of miRNA sequences have been determined in diverse plant species, reports demonstrating the functional roles of miRNAs in the plant response to pathogens are severely limited. Here, Arabidopsis thaliana miRNA844 (miR844) was investigated for its functional role in the defense response to diverse pathogens. Transgenic Arabidopsis plants overexpressing miR844 (35S::miR844) displayed much more severe disease symptoms than the wild-type plants when challenged with the bacterium Pseudomonas syringae pv. tomato DC3000 or the fungus Botrytis cinerea. By contrast, a loss-of-function mir844 mutant showed an enhanced resistance against the pathogens. Although no cleavage was observed at the predicted cleavage site of the putative target mRNA, cytidinephosphate diacylglycerol synthase3 (CDS3), cleavage was observed at 6, 12, 21, or 52 bases upstream of the predicted cleavage site of CDS3 mRNA, and the level of CDS3 mRNA was downregulated by the overexpression of miR844, implying that miR844 influences CDS3 transcript level. To further confirm that the miR844-mediated defense response was due to the decrease in CDS3 mRNA level, the disease response of a CDS3 loss-of-function mutant was analyzed upon pathogen challenge. Increased susceptibility of both cds3 mutant and 35S::miR844 plants to pathogens confirmed that miR844 affected the defense response by downregulating CDS3 mRNA. The expression of miR844 was decreased, and the CDS3 transcript level increased upon pathogen challenge. Taken together, these results provide evidence that downregulation of miR844 and a concomitant increase in CDS3 expression is a defensive response of Arabidopsis to bacteria and fungi.