The extracellular matrix is fundamental in order to maintain normal function in many organs such as the blood vessels, heart, liver, or bones. When organs fail or experience injury, tissue engineering and regenerative medicine elicit the production of constructs resembling the native extracellular matrix, supporting organ restoration and function. In this regard, is it possible to optimize structural characteristics of nanofiber scaffolds obtained by the electrospinning technique? This study aimed to produce partially degraded collagen (gelatin) nanofiber scaffolds, using the electrospinning technique, with optimized parameters rendering different morphological characteristics of nanofibers, as well as assessing whether the resulting scaffolds are suitable to integrate primary human endothelial progenitor cells, obtained from peripheral blood with further in vitro cell expansion. After different assay conditions, the best nanofiber morphology was obtained with the following electrospinning parameters: 15 kV, 0.06 mL/h, 1000 rpm and 12 cm needle-to-collector distance, yielding an average nanofiber thickness of 333 ± 130 nm. Nanofiber scaffolds rendered through such electrospinning conditions were suitable for the integration and proliferation of human endothelial progenitor cells.