AbstractTheMildew Resistance Locus O(MLO) gene family has been investigated in many species. However, there are few studies on chrysanthemumMLOgenes. We report in this study thatCmMLO17inChrysanthemum morifoliumwas upregulated afterAlternaria alternatainfection. Silencing ofCmMLO17by artificial microRNA resulted in reduced susceptibility of chrysanthemum toA. alternatainfection. Genes in the abscisic acid (ABA) and Ca2+signaling pathways were upregulated in theCmMLO17-silenced line R20 compared to the wild-type plants. We speculated thatCmMLO17-silenced plants had a faster and stronger defense response that was mediated by the ABA and Ca2+signaling pathways, resulting in reduced susceptibility of chrysanthemum toA. alternatainfection. In addition, a candidate gene,CmKIC, that may interact with CmMLO17 was discovered by the yeast two-hybrid assay. The interaction between CmMLO17 and CmKIC was confirmed using the yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) analysis. CmMLO17 and CmKIC were both located on the plasma membrane, and CmKIC was also located on the nucleus.CmKICoverexpression increased the susceptibility of chrysanthemum toA. alternata, whereasCmKICsilencing resulted in reduced susceptibility. Therefore, CmMLO17 and CmKIC may work together inC. morifoliumto support the growth ofA. alternata. The results of this study will provide insight into the potential function of MLO and improve the understanding of plant defense responses to necrotrophic pathogens.