Protein is a promising material for fabricating the biocompatible films used in the biomedical fields and food industry. Previously, we successfully prepared a water-insoluble albumin film possessing native albumin properties such as resistance to cell adhesion and drug-binding ability. Here, I quantitatively investigated the conformation of albumin in a film state using circular dichroism (CD) spectroscopy. The albumin film was prepared by crosslinking albumin with ethylene glycol diglycidyl ether (EGDE). CD measurements of albumin films revealed that approximately 70% of the a helical structure was retained after film formation. Albumin molecules in the films acquired high stability. The conformation of albumin was completely retained even after heating at 100 C for 1 h. For comparison, crosslinked albumin film was also prepared using glutaraldehyde (GA). Unlike EGDE-crosslinking, GA-crosslinking induced significant conformational changes in albumin; 46% of the a-helical structure was destroyed in GA-crosslinked albumin films. Cell adhesion studies showed that EGDE-crosslinked albumin film maintained the cell-nonadhesive property inherent in native albumin. This property was lost in GA-crosslinked albumin film, and cells adhesion occurred at a level comparable to that of cell culture dishes. These results indicate that EGDE-crosslinking is a useful method for preparing albumin films in which the native albumin structure and property are retained. The approach described here provides valuable information for creating protein films possessing high functionality. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.