Background <p>In the present study, we investigated the immunotherapeutic and anticancer activities of microRNA-4477a (miR-4477a) as a PD-L1 inhibitor in breast cancer cells (MCF-7).</p> Methods <p>To this end, a series of analytical procedures were conducted, including bioinformatic analysis, RT-PCR analysis, PD-L1 ELISA, in vitro co-culture analysis, cytotoxicity assays, cell migration assays, and colony formation assays, with the objective of determining the anticancer activity of the compound in question.</p> Results <p>The results demonstrated that miR-4477a can bind to three distinct regions of PD-L1 mRNA with high scores (94%, 88% and 80%), effectively targeting and suppressing the crucial regulatory pathways of cancer cells. In vitro studies demonstrated that a 25 nM dose of miR-4477a caused relatively high cytotoxicity in the MCF-7 cell line, suppressed PD-L1 gene expression, and decreased sPD-L1 protein levels, strongly inhibited cell migration, and significantly reduced colony formation. The in vitro co-culture analysis revealed that cancer cells were unable to evade the surveillance and cytotoxic activity of T cells (CD8⁺) due to the blockade of PD-L1 expression by miR-4477a.</p> Conclusions <p>In conclusion, miRNA-4477a has the capacity to regulate immune responses in breast cancer cells and may therefore be a promising candidate for use in cancer immunotherapy as a therapeutic agent.</p> Graphical Abstract <p></p>