The safety evaluation of N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride (HTCC) microspheres as parenteral vaccine adjuvant was carried out in mice, rats, guinea pigs, rabbits. Mouse acute toxicity test showed that they have a high tolerance for over 50 times given clinical dose (5.0 mg/dose). Even high dosage of microspheres was injected in SD rats for many times, no pathological changes were found in their organ. The symptoms of mice in abnormal toxicity test were normal after injection. And the weight of mice increased significantly. In allergy test, no allergic reaction and symptoms were found in guinea pigs. And no fester and suppuration were found in muscle stimulation test. In the hemolysis results, red blood cell did not aggregate. All the tests proved good security of the HTCC microspheres.