This study presents a novel approach to treating hypertrophic scar (HS) using endogenous stimuli-responsive separating microneedles (MNs) loaded with 5-fluorouracil (5-FuA-Pep-MA) prodrug. The MNs are designed to respond to high reactive oxygen species (ROS) levels and overexpression of matrix metalloproteinases (MMPs) in the HS pathological microenvironment. In vivo experiments in female mice demonstrated that the MN tips retained in the tissue provided a slowly sustained drug release. Drug-loaded MNs effectively remodeled the pathological microenvironment by scavenging ROS and consuming MMPs, leading to reduced fibroblast proliferation, collagen deposition, and improved skin fibrosis. RNA sequencing analyses confirmed that drug-loaded MNs reversed skin fibrosis through down-regulation of BCL-2-associated death promoter (BAD) and insulin-like growth factor 1 receptor (IGF1R) pathways, while also regulating inflammatory responses and keratinocyte differentiation. Single-cell RNA sequencing further revealed the central role of interactions between fibroblasts and keratinocytes in HS treatment with drug-loaded MNs. This study highlights the potential therapeutic mechanism of drug-loaded MNs in HS treatment and their broad prospects for clinical application.This study presents a novel approach to treating hypertrophic scar (HS) using endogenous stimuli-responsive separating microneedles (MNs) loaded with 5-fluorouracil (5-FuA-Pep-MA) prodrug. The MNs are designed to respond to high reactive oxygen species (ROS) levels and overexpression of matrix metalloproteinases (MMPs) in the HS pathological microenvironment. In vivo experiments in female mice demonstrated that the MN tips retained in the tissue provided a slowly sustained drug release. Drug-loaded MNs effectively remodeled the pathological microenvironment by scavenging ROS and consuming MMPs, leading to reduced fibroblast proliferation, collagen deposition, and improved skin fibrosis. RNA sequencing analyses confirmed that drug-loaded MNs reversed skin fibrosis through down-regulation of BCL-2-associated death promoter (BAD) and insulin-like growth factor 1 receptor (IGF1R) pathways, while also regulating inflammatory responses and keratinocyte differentiation. Single-cell RNA sequencing further revealed the central role of interactions between fibroblasts and keratinocytes in HS treatment with drug-loaded MNs. This study highlights the potential therapeutic mechanism of drug-loaded MNs in HS treatment and their broad prospects for clinical application.