Subacromial bursa fibrosis are linked to rotator cuff lesion with shoulder stiffness; however, the mechanism underlying this shoulder disorder remain elusive. MicroRNA-29s (miR-29s) are emerging fibrosis inhibitor targeting fibrogenic matrices during tissue fibrosis. This study is aimed to investigate clinical relevance and function of miR-29 signalling to subacromial bursa homeostasis in shoulder stiffness. Subacromial bursa in patients with rotator cuff lesion with or without shoulder stiffness who required open acromioplasty were harvested for assessing fibrosis histopathology using Manson's trichrome staining. Expressions of proinflammatory cytokines, fibrotic matrices, and miR-29s were quantified using RT-PCR and in situ hybridization. Range of motion and pain scores of the stiffness group were higher than those of non-stiffness group. Upregulated proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and fibrotic matrices (collagen 1α1, 3α1, and 4α1) but decreased miR-29a and b expression existed in the stiffness group. Affected tissues exhibited severe fibrotic matrix accumulation, synovial hyperangiogenesis, hyperplasia, and strong miR-29a transcripts. In vitro, IL-1β rather than IL-6 and TNF-α decreased miR-29a expression of subacromial bursa fibroblasts. miR-29a knockdown escalated fibrotic matrix expression, whereas forced miR-29a expression alleviated the IL-1β-induced fibrotic matrix expression. Of interest, miR-29a transgenic mice displayed moderate responses to supraspinatus and infraspinatus tenotomy-induce fibrosis and gait irregularity of affected shoulders. Weak miR-29 signalling causes excessive fibrosis and remodelling in subacromial bursa and ultimately increases the prevalence of shoulder stiffness. This study reveals a new mechanistic underlying shoulder stiffness and highlights that sustained miR-29a potentially ameliorates the severity and function of stiff shoulder