tRNA-derived small RNAs (tsRNAs) have been shown to play regulatory roles in many physiological and pathological processes. However, their roles in hypertrophic scars remain unclear. The present study investigated differentially expressed tsRNAs in human hypertrophic scar fibroblasts and normal skin fibroblasts via high-throughput sequencing. Several dysregulated tsRNAs were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, target prediction, coexpression networks and competing endogenous RNA (ceRNA) networks were evaluated to discover the principal functions of significantly differentially expressed tsRNAs. In total, 67 differentially expressed tsRNAs were detected, of which 27 were upregulated and 40 downregulated in hypertrophic scar fibroblasts. The GO analysis indicated that the dysregulated tsRNAs are associated with numerous biological functions, including 'nervous system development', 'cell adhesion', 'focal adhesion', 'protein binding', 'angiogenesis' and 'actin binding'. KEGG pathway analysis revealed that the most altered pathways include 'Ras signaling pathway', 'Rap1 signaling pathway' and 'cGMP-PKG signaling pathway'. The target genes of the differentially expressed tsRNAs participate in several signaling pathways important for scar formation. The results of RT-qPCR were consistent with those of sequencing. MicroRNA (miR)-29b-1-5p was identified as a target of tsRNA-23678 and was downregulated in hypertrophic scar fibroblasts, constituting a negative regulatory factor for scar formation. Furthermore, tsRNA-23761 acted as a ceRNA and bound to miR-3135b to regulate the expression of miR-3135b targets, including angiotensin-converting enzyme. collectively, these findings reveal that tsRNAs are differentially expressed in human hypertrophic scar fibroblasts, and may contribute to the molecular mechanism and treatment of hypertrophic scars.