The use of 316L stainless steel as an orthopedic bone plate material or as a substrate for coating implies the need to study its properties in a biological medium simulating human body environment. Therefore, the aim of this work is to investigate the microstructural characteristics, corrosion resistance, and mechanical properties of 316L stainless steel in the culture medium and then to check the cytotoxicity of the stainless steel in the presence of human mesenchymal stem cells. The characteristics of the stainless steel were evaluated by studying the microstructure of the samples in untreated state and after soaking in minimum essential medium‐alpha (α‐MEM) for 7 days. The results indicated the formation on the surface of the 316L stainless steel of goethite and deposits composed of Ca and P with porous and amorphous structures, which led to the decrease in the elastic modulus and the hardness of the soaked stainless steel as shown by nanoindentation test. Nevertheless, the hardness and the elastic modulus of the steel after soaking were influenced only for a surface depth of approximately 0.1% of the thickness of the sample. The potentiodynamic polarization curves exhibited that the corrosion resistance of the soaked stainless steel was reduced because of the higher passive current density. However, it should be noticed that the breakdown potential of the steel after soaking was slightly higher, leading to a wider range of passivation. In addition, microscopy examination showed the mesenchymal stem cell adhesion on the materials after 9 days of culture.