Fibrin microthreads are discrete biopolymer fibers, 50-100 μm in diameter, produced from the natural extracellular matrix protein of the provisional matrix that promotes tissue regeneration in the in vivo wound healing environment. The goals of this study were to investigate the feasibility of creating fibrin microthreads containing fibroblast growth factor-2 (FGF-2), and to study the potential of a fibrin matrix to bind signaling proteins known to promote wound healing and regulate cell function in localized cellular microenvironments on scaffold surfaces. FGF-2 was loaded into fibrin microthreads in concentrations ranging from 0 to 200 ng/mL, to investigate the effect of the material on fibroblast attachment, proliferation, cellular outgrowth, and alignment. Although FGF-2-loaded microthreads did not affect fibroblast attachment, they significantly increased cellular outgrowth and proliferation relative to unloaded microthreads. The most pronounced effects were observed at day 7 of cell culture. Further, all of the fibrin microthreads promoted the alignment of fibroblasts and their cytoskeletal components along the long axis of threads, independent of the FGF-2 concentration. Ultimately, we anticipate that these fibrin microthreads will be a promising biopolymer material to promote the regeneration of injured tissues because of their mechanical stability and their matrix signaling capabilities, particularly when loaded with matrix-bound growth factors such as FGF-2.