Background: Integrin β4 (ITGB4) has been shown to enhance the invasiveness of papillary thyroid carcinoma (PTC). In addition, several microRNAs (miRNAs) have been identified as influencing the invasiveness of PTC. Aims: This study aims to explore whether ITGB4 exerts its pro-invasive effects by modulating miRNA expression. Methods and Results: Using RNA sequencing techniques, we found miR-21-5p was significantly upregulated in expression levels following the knockdown and introduction of ITGB4 expression in two PTC cell lines, K1 and TPC-1, respectively. Wound healing and transwell assays indicated miR-21-5p mediates the invasiveness of PTC cells induced by ITGB4. Analysis of the TCGA database revealed a positive correlation between the expression of both ITGB4 and miR-21-5p with increased lymph node metastasis, extra-thyroidal extension, and advanced TNM stages. Mechanistically, a negative correlation between the methylation levels of the miR-21 gene and the expression levels of both ITGB4 and miR-21-5p was identified based on bioinformatic analysis. RT-qPCR and bisulfite sequencing PCR demonstrated the reduction in miR-21-5p expression following ITGB4 knockdown was associated with increased methylation of the miR-21 gene. Treatment with the methyltransferase inhibitor 5-Aza-CdR reversed the methylation changes in the miR-21 gene and substantially restored miR-21-5p expression. Conversely, the induced expression of ITGB4 in TPC-1 cells led to a significant increase in miR-21-5p levels and a decreased methylation of the miR-21 gene. Conclusion: miR-21-5p is identified as a potential downstream miRNA of ITGB4, contributing to the pro-invasive effects associated with ITGB4 in PTC. ITGB4 enhances the expression of miR-21-5p by reducing the methylation levels of the miR-21 gene. The activation of the ITGB4/miR-21-5p axis may provide a molecular basis for the increased invasiveness of PTC.