Composite nano-fibers of poly(caprolactone) (PCL) and gelatin crosslinked with genipin are prepared. The contact angles and mechanical properties of crosslinked PCL-gelatin nanofibers decrease as the gelatin content increases. The proliferation of myoblasts is higher in the crosslinked PCL-gelatin nanofibers than in the PCL nanofibers, and the formation of myotubes is only observed on the crosslinked PCL-gelatin nanofibers. The expression level of myogenin, myosin heavy chain, and troponin T genes is increased as the gelatin content is increased. The results suggest that PCL-gelatin nanofibers crosslinked with genipin can be used as a substrate to modulate proliferation and differentiation of myoblasts, presenting potential applications in muscle tissue engineering.