Surface properties of implanted biomaterials can cause fibrotic tissue reactions by stimulating differentiation of host fibroblasts into contractile myofibroblasts. Silk fibroin (SF) protein has been used as biomaterial in pure and blended form. however, its effect on myofibroblast differentiation remains elusive. We here conjugated SF with lactose using cyanuric chloride as coupling spacer. NMR spectroscopy and the conjugates ability to agglomerate Abrus precatorius agglutinin verified efficient conjugation. Two-dimensional films and three-dimensional scaffolds produced from pure and lactose-conjugated SF solutions were tested as culture substrates for subcutaneous fibroblasts and myofibroblasts. Lactose-conjugated SF substrates mediated higher adhesion, proliferation and viability of fibroblastic cells than pure SF. This SF film composition promotes better attachment of fibroblasts than myofibroblasts. Pro-fibrotic cytokine TGFβ1 was ineffective in inducing fibroblast-to-myofibroblast differentiation on such substrates. Pre-differentiated myofibroblasts lost their contractile phenotype within a few days of being cultured on lactose-conjugated SF. Myofibroblast differentiation was also suppressed by growth in three-dimensional lactose-conjugated SF scaffolds that, however, support population with fibroblasts. We propose that this biomaterial will promote tissue integration without causing a fibrotic host reaction. © 2008 Elsevier Ltd. All rights reserved.