Background: Cigarette smoke exposure (CSE) is a major cause of chronic obstructive pulmonary disease (COPD). The smoke disrupts cell-cell adhesion by inducing epithelial barrier damage to the tight junction (TJ) proteins. Even though the inflammatory mechanism of chemokine (C-C motif) ligand 3 (CCL3) in COPD has gained increasing attention in the research community, however, the underlying signaling pathway, remains unknown. Objectives: To identify the relationship of CCL3 in the pathogenesis of tight junction impairment in COPD and the pathway through which CSE causes damage to TJ in COPD via CCL3, both in vivo and in vitro. Methods: We screened the inflammatory factors in the peripheral blood mononuclear cells (PBMCs) from healthy controls and patients at each GOLD 1-4 stage of chronic obstructive pulmonary disease. RT-PCR, western blot, and ELISA were used to detect the levels of CCL3, ZO-1, and occludin after Cigarette smoke exposure. Immunofluorescence was applied to examine the impairment of the TJs in 16-HBE and A549 cells. The reverse assay was used to detect the effect of a CCR5 antagonist (DAPTA) in COPD. In the CSE-induced COPD mouse model, H&E staining and lung function tests were used to evaluate the pathological and physical states in each group. Immunofluorescence was used to assess the impairment of TJs in each group. ELISA and RT-PCR were used to examine the mRNA or protein expression of CCL3 or miR-4456 in each group. Results: The in vivo and in vitro results showed that CCL3 expression was increased in COPD compared with healthy controls. CCL3 caused significant injury to TJs through its C-C chemokine receptor type 5 (CCR5), while miR-4456 could suppress the effect of CCL3 on TJs by binding to the 3 '-UTR of CCL3. Conclusion: miR-4456/CCL3/CCR5 pathway may be a potential target pathway for the treatment of COPD.