Aphids are serious pests in crop plants. In an effort to identify plant genes controlling resistance against aphids, we have here studied a protease inhibitor, CI2c in barley (Hordeum vulgare L.). The CI2c gene was earlier shown to be upregulated by herbivory of the bird cherry-oat aphid (Rhopalosiphum padi L.) in barley genotypes with moderate resistance against this aphid, but not in susceptible lines. We hypothesized that CI2c contributes to the resistance. To test this idea, cDNA encoding CI2c was overexpressed in barley and bioassays were carried out with R. padi. For comparison, tests were carried out with the green peach aphid (Myzus persicae Sulzer), for which barley is a poor host. The performance of R. padi was not different on the CI2c-overexpressing lines in comparison to controls in test monitoring behavior and fecundity. M. persicae preference was affected as shown in the choice test, this species moved away from control plants, but remained on the CI2c-overexpressing lines. R. padi-induced responses related to defense were repressed in the overexpressing lines as compared to in control plants or the moderately resistant genotypes. A putative susceptibility gene, coding for a β-1,3-glucanase was more strongly induced by aphids in one of the CI2c-overexpressing lines. The results indicate that the CI2c inhibitor in overexpressing lines affects aphid-induced responses by suppressing defense. This is of little consequence to the specialist R.padi, but causes lower non-host resistance towards the generalist M. persicae in barley.