A critical issue in drug development is developing effective, noninvasive delivery routes to the central nervous system (CNS). Major depressive disorder (MDD) is an illness associated with significant morbidity. Even with multiple antidepressant trials, 10–15% of patients continue to experience persistent depressive symptoms. We previously developed an interfering peptide that has antidepressant-like effects in rats when injected directly into the brain. To be clinically viable, it must demonstrate efficacy via a noninvasive administration route. We report here that the interfering peptide designed to disrupt the interaction between the D1 and D2 dopamine receptors can be delivered to relevant brain areas using the Pressurized Olfactory Device (POD), a novel intranasal delivery system developed by Impel NeuroPharma. We validate this delivery method by demonstrating that, at doses⩾ 1.67 nmol/g, the D1–D2 interfering peptide has a significant antidepressant-like effect comparable to that of imipramine in the forced swimming test (FST), a common test for antidepressant efficacy. The antidepressant-like effect of the interfering peptide can be detected for 2 h after intranasal administration. Furthermore, we show that the interfering peptide disrupts the D1–D2 interaction and it can be detected in the prefrontal cortex after intranasal administration. This study provides strong preclinical support for intranasal administration of the D1–D2 interfering peptide as a new treatment option for patients suffering from MDD.