Regenerated silk fibroin films are normally produced by increasing the Silk II structure (beta-sheet content). In the present study, silk fibroin films were prepared by controlling the environmental temperature and humidity, resulting in the formation of silk films with a predominant Silk I structure instead of Silk II structure. Wide angle X-ray diffraction indicated that when the relative humidity was 55%, the silk films prepared were mainly composed of Silk I structure, whereas silk films formed on other relative humidity had a higher Silk II structure. Fourier transform infrared analysis (FTIR) results also conformed that the secondary structure of silk fibroin can be controlled by changing the humidity of the films formed process. Thermal analysis results revealed Silk I structure was a stable crystal, and the degradation peak increased to 320 degrees C, indicating a greater thermal stability of these films formed under the 55% relative humidity conditions. Atomic force microscopy (AFM) results depicted silk fibroin in the fresh solution had many nanospheres existing with 2050 nm diameters and mainly maintained a random coil structure without specific nanostructures. At the same time, it also illustrated the self-assembly process of silk fibroin in the aqueous solution without any human intervention. In addition, this present study also provided additional support for self-assembly mechanism of silk fibroin films formation. (c) 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012