The nasal pathway represents a non-invasive administration route of pharmaceutical agents with local, systemic, and central nervous system action. Intranasal application has a relatively short time to onset of effect and high bioavailability because it avoids hepatic first-pass metabolism. However, sustained delivery is important because drugs can be rapidly eliminated via mucociliary clearance. Protein transduction using arginine-rich cell-penetrating peptides (poly-arginine) shows high delivery efficiency, no cell specificity, and minimal cytotoxicity. We investigated the effect of poly-arginine on protein delivery into the nasal mucosa and brain of mice, and its ability to prolong contact between the delivered molecule and nasal mucosa by preventing mucociliary clearance. Enhanced green fluorescent protein (EGFP) fused to a nine-arginine peptide (EGFP-9R group) or EGFP (EGFP group) was administered once to the bilateral nasal cavities of mice. Histopathological evaluation was conducted for 3–120 h to evaluate side effects, and the number of sneezes was recorded before and after administration. EGFP was detected in cells lining the nasal cavity and their vicinity for 3–96 and 3–24 h in the EGFP-9R and EGFP groups, respectively. EGFP was detected in the brain at 3–96 h in the EGFP-9R group but not in the EGFP group. Nasal symptoms and histopathological assessment revealed no deterioration in either group. These results suggest that protein transduction using poly-arginine can deliver therapeutically relevant molecules for allergic rhinitis, and can be applied for olfactory disturbance and other central nervous system diseases. Further research is necessary to establish therapy protocols using this technique.