Silk fibroin-chitosan (SFCS) scaffold is a naturally derived biocompatible matrix with potential reconstructive surgical applications. In this study, human adipose-derived mesenchymal stem cells (ASCs) were seeded on SFCS scaffolds and cell attachment was characterized by fluorescence, confocal, time-lapse, atomic force, and scanning electron microscopy (SEM) studies. Adhesion of ASCs on SFCS was 39.4 + or – 4.8% at 15 min, increasing to 92.8+/-1.5% at 120 min. ASC adhered at regions of architectural complexity and infiltrate into three-dimensional scaffold. Time-lapse confocal studies indicated a mean ASC speed on SFCS of 18.47+/-2.7 microm h(-1) and a mean persistence time of 41.4 + or – 9.3 min over a 2.75 h study period. Cytokinetic and SEM studies demonstrated ASC-ASC interaction via microvillus extensions. The apparent elastic modulus was significantly higher (p<0.0001) for ASCs seeded on SFCS (69.0 + or - 9.0 kPa) than on glass (6.1 + or - 0.4 kPa). Also, cytoskeleton F-actin fiber density was higher (p<0.05) for ASC seeded on SFCS (0.42 + or - 0.02 fibers microm(-1)) than on glass-seeded controls (0.24 + or - 0.03 fibers microm(-1)). Hence, SFCS scaffold facilitates mesenchymal stem cell attachment, migration, three-dimensional infiltration, and cell-cell interaction. This study showed the potential use of SFCS as a local carrier for autologous stem cells for reconstructive surgery application. ©2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.