Chitosan is a biopolymer with bactericidal/bacteriostatic effect, biocompatible and biodegradable. It has been used in tissue engineering to replace tissues partially or completely by releasing bioactive materials or influencing cell growth, usually in regenerative medicine and dentistry. The aim of this study was to evaluate the cytotoxic and anti-inflammatory effect of chitosan alone or with hemostatic gelatin (Spongostand®) in cultures of human pulp cells (HPC), human gingival fibroblasts (HGF) and mouse pre-osteoblasts (MC3T3-E1, ATCC). HPC and HGF were isolated from patients. Cells were subcultured in DMEM. Chitosan was inoculated at different concentrations (0-0.5%) and hemostatic gelatins impregnated with chitosan (0.19%) were placed directly in the presence of cells and incubated for 24 hours. Cell viability was determined by MTT method and mean cytotoxic concentration (CC50) was calculated from the dose-response curve. Anti-inflammatory effect was calculated from the in vitro gingivitis model induced with interleukin 1beta (IL-1β) in HGF and protein detection. The data were subjected to Shapiro-Wilk, Kruskal-Wallis and Mann-Whitney tests. Experiments were performed in triplicate of three independent assays. Cell viability of HPC, HGF and MC3T3-E1 in contact with chitosan decreased significantly (p<0.05). The HPC were the most sensitive (CC50= 0.18%), followed by HGF (CC50= 0.18%) and MC3T3-E1 (CC50= 0.19%). The cytotoxicity of gelatins impregnated with chitosan decreased cell viability of HGF and HPC by 11% and 5%, respectively. The proinflammatory effect was reduced significantly in the gingivitis model. To conclude, chitosan induces moderate cytotoxic effects alone or with hemostatic gelatin at 0.19%, in dose-dependent manner, with anti-inflammatory effects on human gingival fibroblasts. The use of chitosan as a biomaterial can be an excellent choice for use in regenerative dentistry.