Cytocompatibility of implant material is dependent on the processed surface topography. This study aimed to characterize the milling surface topography features and to assess the cytotoxicity of PEEK processed using end milling technology. Based on the Taguchi method, end milling experiments have been conducted on PEEK to characterize surface topographies induced by various milling conditions. Machined surface defects and 3-Dimensional surface topography parameters such as surface average height Sa, the tenpoint height of surface Sz, Skewness Ssk, and Kurtosis Sku were characterized. The relationships between the cutting technological parameters and surface topography parameters were established by developing a linear regression model. On the other hand, the effect of the machined surface topography on the viability of fibroblasts was investigated by using cells from a mouse fibroblast L929 and the agar overlay test, MTT test. The experimental result indicated that feed marks, scratch marks, budlike protuberances, the adhered surface particles are the main forms of the surface defect in the end milling of PEEK. The axial and radial depths of cut are the two most critical factors to affect surface topography parameters. Agar overlay tests produced no evidence of cell damage caused by PEEK, and a conclusion can be drawn that PEEK has no cytotoxicity to L929 cell. MTT test results indicated that milling-induced surface topography features exerted specific toxicity on L929 cells, and a statistical difference between the cell's viability of the test samples is observable. By correlation analysis, the correlations order of the viability of fibroblasts L929 with the machined surface roughness parameters were Ssk, Sa, Sku and Sz in large to small. It is concluded that end milling processing significantly affects surface topography which in turn influences the L929 fibroblast cellular response.A significant decrease of L929 viability started with the surface roughness of Sa = 1.18 μm, and surface roughness characterized by Ssk = 0.011 (namely, Ssk closed to zero) also 2 / 23 allowed to achieve the highest possible cells viability.