Abstract: Objective To detect the biocompatibility of porous gelatin microspheres in adipose-derived stem cells (ADSCs) of rats and provide theoretical foundation for further experiments. Methods ADSCs were isolated from rats and then were induced to differentiate into adipogenic, chondrogenic and osteogenic lineages. The proliferation and toxicity were assessed using MTT assay at 1 d, 3 d and 5 d respectively after ADSCs were incorporated into the microspheres. Laser confocal microscopy was used to observe the morphology and proliferation of cells on the scaffolds. Results Scanning electron microscopy image showed that majority of the microspheres diameters were 100-400μm and the pore diameters were 20-90μm. Most of ADSCs grew well in a spindle morphology and adipogenic, chondrogenic and osteogenic differentiation were induced successfully. There was no significant difference in OD value between the leaching solution group and the normal stem cell culture medium group. OD value of the ADSCs incorporated into the microspheres group was significantly higher than that of the normal cell culture group. Laser confocal microscopy examination showed that the cells on the microspheres had a rapid proliferation after 5 days. Conclusion Gelatin porous microspheres can promote the cells proliferation with nice biocompatibility, which lays foundations for further biological experiments.