Pelvic fracture urethral distraction defect (PFUDD) seriously affects the quality of life of patients. At present, there are few effective drug treatments available for PFUDD‑induced urethral stricture, which is associated with fibrosis and scar formation in urethra lumen. Emerging evidence suggests that microRNAs (miRNAs/miRs) may be involved in the regulation of fibrosis, and analysis of miRNA expression profiles in urethral scar and normal urethra tissues may therefore benefit the discovery of novel treatments for urethral stricture with micro invasive procedures. In the present study, miRNA sequencing and quantitative polymerase chain reaction (qPCR) validation using paired scar and normal tissues from patients with PFUDD, and functional analysis of the miRNAs involved in the fibrosis associated signaling pathway was performed. A total of 94 differentially expressed miRNAs were identified in the scar tissue of patients with PFUDD. Among them, 26 miRNAs had significantly altered expression in the scar tissue compared with the normal tissue from the same patient. qPCR validation confirmed that miR‑129‑5p was overexpressed in scar tissue. The TGF‑β pathway‑associated functions of a total of 5 miRNAs (hsa‑miR‑129‑5p, hsa‑miR‑135a‑5p, hsa‑miR‑363‑3p, hsa‑miR‑6720‑3p and hsa‑miR‑9‑5p) were further analyzed, as well as their key molecular targets and functional mechanisms in signaling regulation. To conclude the miRNA sequencing indicated a significantly altered expression of hsa‑miR‑129‑5p, hsa‑miR‑135a‑5p, hsa‑miR‑363‑3p, hsa‑miR‑6720‑3p and hsa‑miR‑9‑5p in patients with PFUDD. These miRNAs and their potential target genes were associated with fibrosis in several diseases, and the data from the present study may help explore potential miRNA targets for future precision treatments for urethral stricture.