The objective of this study was to characterize the surface oxide film on nickel-free austenitic stainless steel, Fe-24Cr-2Mo-1N in mass% , with nitrogen absorption located in various environments, to estimate the reconstruction of the surface oxide film in the human body. The specimens were metallographically polished in deionized water, autoclaved after polishing, immersed in Hanks' solution for 7 days after polishing and autoclaving, immersed in Eagle's minimum essential medium containing fetal bovine serum for 7 days after polishing and autoclaving, and incubated with L929 fibroblasts for 7 days after polishing and autoclaving. L929 fibroblasts cultured on the specimen were removed with a flow of deionized water before surface analysis. X-ray photoelectron spectroscopy was performed to determine the composition of the surface oxide film and substrate and the thickness of the surface oxide film. The depth profiles of elements in the surface region were also characterized using Auger electron spectroscopy in combination with argon-ion-sputtering. The surface oxide film on the nickel-free austenitic stainless steel with nitrogen absorption, Fe-24Cr-2Mo-1N, polished mechanically in deionized water, consisted of iron and chromium oxides containing small amount of molybdenum oxide. A large amount of carbon, nitrogen, oxygen, and sulfur originating from proteins was detected after immersion in the medium and incubation with the cells. Sulfur existed as sulfide or sulfite. Calcium phosphate was formed on the surface oxide film after immersion in the Hanks' solution and medium and incubation with the cells.