While substrate topography influences cell behavior, RNA interference (RNAi) has also emerged as a potent method for understanding and directing cell fate. However, the effects of substrate topography on RNAi remain poorly understood. Here, we report the influence of nanofiber architecture on siRNA-mediated gene-silencing in human somatic and stem cells. The respective model cells, human dermal fibroblasts (HDFs) and mesenchymal stem cells (MSCs), were cultured onto aligned or randomly oriented electrospun poly(epsilon-caprolactone) fibers of different average diameters (300 nm, 700 nm and 1.3 mu m). In HDFs, decreasing fiber diameter from 1.3 mu m to 300 nm improved Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Collagen-I silencing efficiencies by similar to 3.8 and similar to 4.4 folds respectively (p < 0.05) while the effective siRNA uptake pathway was altered from clathrin-dependent endocytosis to macro-pinocytosis. In MSCs, aligned, fibers generated significantly higher level of gene silencing of RE-1 silencing transcription factor (REST) and green fluorescent protein (GFP) (similar to 1.6 and similar to 1.5 folds respectively, p < 0.05), than randomly-oriented fibers. Aligned fiber topography facilitated functional siRNA uptake through clathrin-mediated endocytosis and membrane fusion. Taken together, our results demonstrated a promising role of three-dimensional fibrous scaffolds in modulating siRNA-mediated gene-silencing and established the critical synergistic role of these substrates in modulating cellular behavior by RNAi. (C) 2014 Elsevier Ltd. All rights reserved.