Hypertrophic scar (HS) is a dermal fibro-proliferative disorder result from abnormal wound healing after skin injury. MicroRNA-9-5p (miR-9-5p) has been reported to be upregulated and closely related to collagen proteins in human dermal fibroblasts. However, the correlation and possible mechanism between miR-9-5p and HS require further investigation. The expressions of miR-9-5p in HS tissues and HS fibroblasts were detected by quantitative real-time PCR (RT-qPCR). The expression level of peroxisome proliferator-activated receptor β (PPARβ) was measured by RT-qPCR assay. The protein levels of PPARβ, α-SMA, Vimentin, COL1A, cyclin D1, bcl-2, and bax were detected by western blot assay. The effect of miR-9-5p and PPARβ on HS fibroblasts proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) and flow cytometry assays. The interaction between miR-9-5p and PPARβ was predicted by TargetScan, and then confirmed by dual-luciferase reporter assay. MiR-9-5p expression was downregulated in HS tissues and HS fibroblasts. MiR-9-5p inhibited the levels of extracellular matrix-associated genes (α-SMA, Vimentin, COL1A) in HS fibroblasts. MiR-9-5p repressed proliferation and induced apoptosis of HS fibroblasts. PPARβ is a target gene of miR-9-5p. The silencing of PPARβ expression hindered proliferation and expedited apoptosis of HS fibroblasts. MiR-9-5p suppressed proliferation and promoted apoptosis of HS fibroblasts by targeting PPARβ. In this paper, we firstly disclosed that miR-9-5p hampered extracellular matrix deposition and proliferation, and induced apoptosis by targeting PPARβ in HS fibroblasts. Our findings provided a new role of miR-9-5p/PPARβ in the occurrence and development of HS fibroblasts, promising a new target for HS. © 2020. Published by The Company of Biologists Ltd.