We investigated proteome and the involvement of barley MPK3 in response to flagellin peptide flg22, a well-known bacterial elicitor. We show that MPK3 knock out lines of barley (Hv MPK3 KO) exhibit constitutive downregulation of defence related proteins such as PR proteins belonging to thaumatin family and chitinases. Further proteomic analyses showed, that the same protein families were less prone to flagellin elicitation in MPK3 KO plants compared to wild types. These results positively correlated with chitinase activity analyses. Differential proteome also indicated possible root growth defects in the mutant plants, which was corroborated with early root growth retardation and altered short- and mid-term reactions to flg22 in terms of radial root expansion and root hair emergence.