Mitogen-activated protein kinases (MAPKs) are fundamental components of the plant immune system. MAPK cascades are activated by microbial elicitors and effectors, such as microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs). The activation of MAPKs plays essential roles in early and late responses associated with plant defense and innate immunity. In the present experiment, we investigated the role of HvMPK3 in the resistance of barley (Hordeum vulgare) against Fusarium graminearum. The proteomes of TALEN-induced MPK3 knock-out lines of barley (HvMPK3 KO) were compared with wild types 24 h after the treatment with F. graminearum. Results explain the resistance of HvMPK KO lines to the pathogen.