Objective <p>To investigate the differential expression of microRNA-144-3p in endometrial cells exposed to copper ions in vitro. The specific mechanism by which microRNA-144-3p is involved in Cu²⁺-induced damage to the human endometrial epithelial cells (HEECs) was explored.</p> Methods <p>HEECs were cultured in copper-containing culture medium to simulate changes in the endometrium after copper intrauterine device (Cu-IUD) implantation. Reverse transcription quantitative PCR (RT-qPCR) was used to detect the differential expression of <i>miR-144-3p</i> in HEECs after Cu²⁺ treatment. MiRNAs, siRNAs and related inhibitors were used to treat HEECs. The expression levels of related downstream genes were then analyzed by RT-qPCR, Western blotting and immunofluorescence to explore the specific mechanism involved.</p> Results <p><i>MiR-144-3p</i> was significantly upregulated in the Cu²⁺-treated HEECs. The expression of P-NF-κB, MMP9, TGF-β3 and P-SMAD3 was significantly decreased in HEECs treated with 10&#xa0;μg/mL Cu²⁺. <i>MiR-144-3p</i> regulated the expression of metallothionein 1A (MT1A) and thrombospondin-1 (THBS-1) in Cu²⁺-treated HEECs. The expression of P-NF-κB can be regulated by MT1A, and an inhibitor of P-NF-κB can significantly reduce the expression of MMP9 in Cu²⁺-treated HEECs. The expression of TGF-β3 can be regulated by THBS-1, and a TGF-β3 inhibitor can significantly reduce the expression of SMAD3 in Cu²⁺-treated HEECs. The proliferative capacity of HEECs treated with MMP9 or SMAD3 inhibitors was significantly reduced.</p> Conclusions <p>The increased Cu²⁺ concentration led to the upregulation of <i>miR-144-3p</i>, further reducing the expression levels of its target genes (MT1A and THBS-1), which in turn downregulated the expression of NF-κB, MMP9, TGF-β3 and SMAD3, ultimately leading to increased endometrial cell damage and decreased cell proliferation.</p>