Objective To compare the characteristics of gelatin microspheres crossl inked by glutaraldehyde (GA) or geni pin (GP). Methods Gelatin microspheres, prepared by the improved emulsified cold-condensation method, were crossl inked by GP and GA, respectively. After being dispersed in PBS, two kinds of microspheres with 60% degree of cross l inking were compared in terms of morphology, swell ing and degrading properties. rhBMP-2 were loaded into the GP and GAmicrospheres, and the encapsulation rate, drug loading and releasing capacity were measured; 100%, 50% and 25% leaching l iquid of GP and GA microspheres were respectively cultured with rat osteoblast (DMEM group as the control), and cell prol iferation was measured by MTT method to grade the cell cytotoxicity. Results GP and GA microspheres were both spherical with the diameters of (78 ± 18) μm and (65 ± 10) μm, and there were no difference between both microspheres in drug loading and encapsulation rate. But, GP microspheres, with long degrading period (28 days) compared to GA microspheres (21 days), had better dispersibil ity, and swell ing rate (89.0% ± 4.8%), the percentage of cumulative drug releasing at 10 days (78.80% ± 4.96%) were both lower than GA microsphere (118.0% ± 7.6%, 90.50% ± 5.12%). The percentages of drug loading of GP and GA were (921 ± 73) and (965 ± 62) ng/g, and the encapsulation rates were 88.5% ± 2.1% and 89.7% ± 1.8%; showing no significant difference (P gt; 0.05). The cell cytotoxicity of 100%, 50% and 25% leaching l iquid of GP microspheres was all at the level I, but leaching l iquid of GA microspheres with corresponding concentration were at the levels of III, III and II. Conclusion GP crossl inked gelatin microspheres are superior to GA crossl inked gelatin microspheres and can be widely used in tissue engineering field.