Problems associated with current bone substitutes led to an increased interest in tissue engineered surrogates with properties similar to natural bones. Commonly used materials lack the ultimate mechanical properties. In this study, we examined the influence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as a crosslinking agent on the strength of collagen fibers. Collagen fibers are crosslinked with EDC, or EDC/N-hydroxysulfosuccinimide (Sulfo-NHS) in water or EDC in acetone. After which fibers are mineralized using calcium chloride and potassium phosphate. The mechanical properties of the treated fibers are measured using tensile testing. In addition, the effect of crosslinking on cellular behavior was tested by culturing bone marrow mesenchymal stem cells (BMMSCs) on modified fibers. We found that the mechanical properties of non-mineralized and mineralized collagen fibers are significantly affected by the crosslinking method. The tensile strength of single fibers is greatly improved by crosslinking with EDC in acetone. BMMSCs were found to attach and spread well on modified fibers confirming biocompatibility. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 2507–2514, 2013.