A simple one-step approach to electrospin Type I collagen in the presence of the chemical crosslinking agents 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) has been developed to generate water-insoluble collagen nanofiber scaffolds without the need for post-crosslinking. SEM images indicate that fibrous surface morphology of collagen scaffolds was well retained after the in situ crosslinking process and following water treatment. The resultant collagen demonstrated a similar uniaxial tensile behavior of native tissue in mechanical testing.