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The aim of this work was to study the cytotoxicity of Ti-6Al-4V-5Cu(TC4-Cu)alloy for dental applications and evaluate the cell viability of different fabricated TC4-Cu alloys in contact with MC3T3-E1 cells in vitro.Ti-6Al-4V(TC4)alloy was used as a negative control to evaluate the cytotoxicity level of TC4-Cu alloy so as to provide basic support for the den-tal clinical application.Control group TC4 and experimental group TC4-Cu with different fabrications were incubated in the cell culture medium.The absorbance value of mouse osteoblast MC3T3-E1 cells was detected by CCK-8 assay after 1,3 and 7 days,respectively.The relative proliferation rate of cells was calculated,and then the toxicity level was valued for each group.Cell morphology on the surface was also studied by observing the cytoskeleton through F-actin filament stain-ing.The experimental results showed that the absorbance values for the experimental and the negative control groups were not same at different time points.Compared with different fabricated TC4-Cu alloys,the annealing-TC4-Cu alloy showed much better biocompatibility.The mouse osteoblast MC3T3-E1 cells cultured with annealing-TC4-Cu,rolling-TC4-Cu and solution+aging-TC4-Cu have no toxic effects,and these alloys could promote the proliferation of mouse osteoblasts.