Keloids are induced by skin injuries such as surgeries, skin piercings, burns, and trauma. The intra-lesional injection of 5-fluorouracil (5-FU) is a promising therapy to treat keloid. However, local 5-FU injections have caused several side effects such as pain at administration and hyperpigmentation. This study suggests a safer and more effective 5-FU delivery system. We used microneedles to treat keloid because this method has the feasibility of self-administration without pain. In this study, 5-FU-loaded carboxymethyl chitosan (CMC) nanoparticles were prepared and characterized by various analytical methods and then coated on stainless solid microneedles. The blank CMC nanoparticles caused an increase in cell viability on human normal fibroblasts to 150%. In particular, the 5-FU-loaded CMC nanoparticles showed a significant inhibitory effect on the human keloid fibroblast to 16%. The intercellular uptake of the 5-FU-loaded CMC nanoparticles was observed on both human normal and keloid fibroblasts by using a confocal microscope. In addition, it was found that the nanoparticles showed an inhibition of TGF-beta 1 by ELISA. For topical drug delivery, it was confirmed that the nanoparticles coated onto the microneedles were dissolved and diffused at the administration site in the porcine dorsal skin model. According to these results, the suggested microneedle-mediated drug delivery system not only inhibits the human keloid fibroblasts by delivering drugs effectively into the keloids but also has the feasibility to self-administer without pain. Therefore, this new system including 5-FU-loaded CMC nanoparticles and microneedles has the potential to treat keloid scars.