Aim: To investigate the enhancing effects of chitosan with or without enhancers on nasal recombinant hirudin-2 (rHV2) delivery in vitro and in vivo, and to evaluate the ciliotoxicity of these formulations. Methods: The permeation-enhancingeffect of various chitosan formulations was estimated by using the permeation coefficient of fluorescein isothiocyanate recombinant hirudin-2 (FITC-rHV2) across the excited rabbit nasal epithelium in vitro. The effect was further evaluated by measuring the blood concentration level after nasal absorption of FITC-rHV2 in rats. The mucosal ciliotoxicity of different formulations was evaluated with an in situ toad palate model. Results: Chitosan at a concentration of 0.5% with or without various enhancers significantly increased the permeability coefficient(P) and relative bioavailability (Fr) of FITC-rHV2 compared with the blank control.The addition of 1% sodium dodecylsulfate, 5% Brij35, 5% Tween 80, 1.5% menthol,1% glycyrrhizic acid monoammonium salt (GAM) or 4% Azone into the 0.5%chitosan solution resulted in a further increase in absorption (P＜0.05) compared with 0.5% chitosan alone. But co-administration of chitosan with 5% hydroxylpropyl-beta-cyclodextrin (HP-β-CD), 5% lecithin or 0.1% ethylenediamine tetraacetic acid (EDTA) was not more effective than using the 0.5% chitosan solution alone.Chitosan alone and with 5% HP-β-CD, 0.1% EDTA, 1% GAM or 5%Tween80 was relatively less ciliotoxic. Conclusion: Chitosan with or without some enhancers was able to effectively promote the nasal absorption of recombinant hirudin, while not resulting in severe mucosal ciliotoxicity. A chitosan formulation system would be a useful approach for the nasal delivery of recombinant hirudin.