Thionins are antimicrobial peptides that are involved in plant defence. Here, we present an in-depth analysis of the role of rice thionin genes in defence responses against two root pathogens: the root-knot nematode Meloidogyne graminicola and the oomycete Pythium graminicola. The expression of rice thionin genes was observed to be differentially regulated by defence-related hormones, whereas all analysed genes were consistently down-regulated in M.graminicola-induced galls, at least until 7days post-inoculation (dpi). Transgenic lines of Oryza sativa cv. Nipponbare overproducing OsTHI7 revealed decreased susceptibility to M.graminicola infection and P.graminicola colonization. Taken together, these results demonstrate the role of rice thionin genes in defence against two of the most damaging root pathogens attacking rice.