A significant challenge remains to protect protein drugs from inactivation during production, storage, and use. In the present study, the stabilization and release of horseradish peroxidase (HRP) in silk films was investigated. Water-insoluble silk films were prepared under mild aqueous conditions, maintaining the activity of the entrapped enzyme. Depending on film processing and post-processing conditions, HRP retained more than 90% of the initial activity at 4 degrees C, room temperature and 37 degrees C over two months. The stability of protein drugs in silk films is attributed to intermolecular interactions between the silk and the enzymes, based on Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The unique structural feature of silk molecules, periodic hydrophobic-hydrophilic domains, enabled strong interactions with proteins. The entrapped protein was present in two states, untrapped active and trapped inactive forms. The ratio between the two forms varied according to processing conditions. Proteolytic degradation and dissolution of the silk films resulted in the release of the bound enzyme which was otherwise not released by diffusion; enzyme recovered full activity upon release. There was a linear relationship between silk degradation/dissolution and the release of entrapped enzyme. Modifying the secondary structure of the silk matrix and the interactions with the non-crystalline domains resulted in control of the film degradation or dissolution rate, and therefore the release rate of the entrapped enzyme. Based on the above results, silk materials are an intriguing carrier for proteins in terms of both retention of activity and controllable release kinetics from the films.