Pathological scars, including hypertrophic scars (HTSs) and keloids, are abnormal wound healing responses, and current treatments are often unsatisfactory. Intralesional multi-injection of 5-fluorouracil (5-Fu), a commonly used approach, has raised great concerns due to its short retention time and adverse effects for anti-scar treatments. To address this, we developed a novel formulation of 5-Fu for scar management, carrier-free pure 5-Fu nanoparticles (nano 5-Fu), prepared by a super-stable pure-nanomedicine formulation technology. In vitro tests on keloid fibroblasts and human umbilical vein endothelial cells (HUVECs) showed that nano 5-Fu possessed superior ability to inactivate these cells compared to free 5-Fu. In vivo retention tests on rabbit HTSs and rat normal skin demonstrated that nano 5-Fu could coexist in soft tissues for a longer time, resulting in a more effective dosage in scar lesions. In vivo results on rabbit scars indicated that nano 5-Fu can lead to a significant reduction in scar elevation index (SEI), collagen fiber deposition and microvessels for skin remodeling. Importantly, the results of the first-in-human trial demonstrated that nano 5-Fu could decrease scar height and vascularity with excellent and stable efficacy as well as less topical side effects. Peripheral blood analyses demonstrated the high systemic safety of nano 5-Fu intralesional injection in HTSs. The carrier-free 5-Fu nanoparticle-mediated “domestication” therapy, featuring low dosage, outstanding efficacy and safety, represents a promising strategy for pathological scar treatment.