Fusarium head blight (FHB) caused by Fusarium graminearum (Fg) severely affects cereal crops, especially wheat and barley. FHB results in significant yield loss, reduces grain quality and contaminates grains with mycotoxin. The development of FHB-resistant cereal cultivars can be expedited through CRISPR gene editing. The Arabidopsis ethylene insensitive 2 (AtEIN2) plays a key role in ethylene signaling pathway and is critical for monitoring plant growth and defense responses. RNAi down-regulation of the wheat homolog TaEIN2 has been shown to enhance wheat FHB resistance. Here we generated site-specific mutations in AtEIN2 by CRISPR-editing. Detached inflorescence infection assays revealed that AtEIN2 knock-out (KO) mutants displayed enhanced Fg resistance and substantially reduced Fg spore production in planta. Gene expression profiling of defense genes revealed that impairment of AtEIN2 resulted in down-regulation of the ethylene signaling pathway while the salicylic acid signaling pathway was unaffected. Complementation of AtEIN2-KO plants with a barley orthologue, HvEIN2, restored Fg susceptibility, indicating that HvEIN2 is functionally equivalent to its Arabidopsis counterpart and, hence, may have a similar role in conditioning barley Fg susceptibility. These results provide insight into the defense role of EIN2 and a molecular and functional foundation for manipulating HvEIN2 to enhance FHB resistance in barley. •Arabidopsis AtEIN2-KO mutants were produced by CRISPR-gene editing.•Arabidopsis AtEIN2-KO mutants were highly resistant to F. graminearum infection.•AtEIN2-KO mutant complemented with barley HvEIN2 regained Fg susceptibility.•New insights on the involvement of ethylene signaling in Fg-host interaction.