Ustilago esculenta, resembling a fungal endophyte in Zizania latifolia, inhibits the host plant flowering and induces the host stems to swell and form edible galls. It is well believed that when and how the fungus infects and proliferates in the host plants during the host development is of importance in the edible gall formation. Mitogen-activated protein kinases (MAPKs) have been found to play an important role in sensing environment cues and regulating infection. Two MAPK genes UeKpp2 and UeKpp6 from U. esculenta were cloned and suggested to be involved in the Fus3/Kss1 pathway by a phylogenetic analysis with the neighbor-joining method. Quantitative RT-PCR (qRT-PCR) analyses indicated that expression of UeKpp2 and UeKpp6 were induced during mating and infection processes, and their expression patterns displayed differentially under different carbon and nitrogen sources. In addition, subcellular localization of UeKpp2 or UeKpp6 fused with the reporter green fluoresce protein was observed by confocal laser scanning microscope, and yeast two-hybrid assays were carried out. Results showed that both UeKpp2 and UeKpp6 were located in cytoplasm and interacted with UePrf1, indicating their involvement in hyphal growth and host-pathogen regulation. Only UeKpp2 but not UeKpp6 interacted with the upstream MAPK kinase UeFuz7, implying an additional MAPK pathway, in which UeKpp6 involved, existed.