Little is known about plant-parasitic animal-derived pathogen-associated molecular pattern (PAMP)/ pattern-recognition receptor (PRR) pairs. Additionally, mitochondrial proteins have not previously been reported to be secreted into hosts by pathogens. Here, it is found that the Meloidogyne javanica elongation factor thermo unstable (EF-Tu) (MjEF-Tu) located in the nematode mitochondria is up-regulated and secreted into the host plant during nematode parasitism. MjEF-Tu interacts with the PRR Arabidopsis thaliana EF-Tu receptor (AtEFR), triggering the plant hallmark defence responses mediated by AtEFR. An 18-aa sequence (Nelf18) in the N terminus of the nematode EF-Tu contributes to the immunogenic activity. M. javanica water extract and mitochondrial extract also induce plant immunity sensed by AtEFR, owing to the presence of MjEF-Tu. In addition, Nelf18 enhances plant resistance to nematode, virus, and bacterial infections depending on AtEFR. These findings first demonstrate that mitochondrial proteins from pathogens can be secreted into hosts and function as a cross-kingdom signal and identified the first plant-parasitic animal-derived proteinaceous PAMP/PRR pair, providing novel insights into host-pathogen interactions.