It has been suggested that supraosseous perforation requires materials such as resin, glass ionomer, and mineral trioxide aggregate (MTA). Since these materials would be in contact with gingival connective tissue, cellular responses to these materials need to be evaluated before clinical use. Materials and Methods: To investigate the biological responses to glass ionomer, resin, and MTA in human gingival fibroblasts by scanning electron microscopy and MTT assay. Results: In scanning electron microscopy analysis, human gingival fibroblasts appeared flat and spread out across the entire glass surface. Cells attached poorly to the amalgam and IRM surfaces and showed round or amorphous morphologies. On resin and glass ionomer surfaces, cells attached well and spread out extensively over the entire surfaces. Cells showed polygonal shapes with filopodial extensions indicative of cell spreading. On MTA surfaces, cells also spread out polygonally over entire surfaces, and cell projections connecting cells were visible. In MTT assay, there was a significant difference among glass and the three other test materials (p<0.05). Cell proliferation rates of resin, glass ionomer, and MTA were 81%, 83%, and 79%, respectively, compared to glass. However, there was no significant difference among the three test materials. Conclusion: These results suggest that resin, glass ionomer, and MTA have good cytocompatibility with human gingival fibroblasts and can be used for the treatment of subgingival perforation or subgingival defects.