Silk fibroin (SF) from silkworms has been widely studied as a biomaterial. The degradation behavior of silk biomaterials is important for medical applications, but few studies have examined long-term degradation behavior in vivo. In this study, we investigated the degradation behavior of SF membranes in vitro and in vivo. For the in vitro assay, we observed degradation of silk membranes in phosphate buffered saline, culture media, and an enzyme (proteinase K) solution. In the proteinase K solution, 80% of the silk membranes degraded within 10 days. Silk membranes exhibited no cytotoxicity toward L929 cells and rat tissues. To investigate the degradation of silk membranes in vivo, they were implanted subcutaneously in rats and harvested 19 months after surgery. Scanning electron microscopy imaging and histological analysis of silk membrane explants showed that they broke into several pieces after 16 months. Results show that silk membranes are biocompatible and display excellent long-term degradation behavior when used as biomaterials. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.