Objective To explore how miR-34a-5p regulates insulin-like growth factor Ⅱ mRNA-binding protein 3 (IMP3) and programmed death ligand-1 (PD-L1) expression and its influence on the biological behavior of breast cancer cells. Methods We analyzed IMP3 and PD-L1 expression in breast cancer and normal tissues using the Human Protein Atlas and GEPIA, and patient prognosis and survival using The Cancer Genome Atlas and GTEx project. miR-34a-5p, IMP3, and PD-L1 mRNA levels, IMP3 and PD-L1 protein levels were detected in breast cancer and adjacent normal tissues by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Correlations between miR-34a-5p and IMP3 and PD-L1 mRNAs were analyzed by Pearson’ s test. Immunohistochemistry was used to detect IMP3, PD-L1, and leukocyte differentiation antigen 44 variant 6 (CD44v6) expression in breast cancer and normal tissues, and the relationships between IMP3 and PD-L1 expression and clinicopathological parameters were analyzed. Target gene prediction and dual luciferase reporter assay were used to verify the targeted regulation of miR-34a-5p on IMP3 and PD-L1. MCF7 cells were divided into control group, miR-NC group, miR-34a-5p group, miR-NC+pcDNA-NC group, miR-NC+IMP3 group, miR-NC+PD-L1 group, miR-34a-5p+pcDNA-NC group, miR-34a-5p+IMP3 group, and miR-34a-5p+PD-L1 group. Cell proliferation, migration, and invasion were determined by MTT, colony-formation, scratch, and Transwell chamber tests, respectively. Expression levels of IMP3, PD-L1, CD44v6, matrix metalloproteinase-2 (MMP-2), MMP-9, and E-cadherin were detected by Western blot. Results The biological databases showed that higher expression levels of IMP3 and PD-L1 in breast cancer tissues were associated with shorter survival (P<0. 05). Expression levels of miR-34a-5p were significantly lower while expression levels of IMP3 and PD-L1 mRNAs and proteins were significantly higher in breast cancer tissues than in normal tissues. miR-34a-5p expression was negatively correlated with IMP3 and PD-L1 mRNAs, respectively (P<0. 05). Immunohistochemistry showed positive expression of IMP3, PD-L1, and CD44v6 in breast cancer, with higher IMP3 and PD-L1 expression associated with later TNM stage, lower degree of tumor differentiation, and increased risks of lymph node and distant metastases (P<0. 05). Dual luciferase assay verified the targeted regulation of miR-34a-5p on IMP3 and PD-L1. The cell viability, number of clones formed, scratch-healing rate, cell invasion, and expression levels of IMP3, PD-L1, CD44v6, MMP-2, and MMP-9 protein were all significantly lower and E-cadherin expression was significantly higher in the miR-34a-5p group compared with the control group and miR-NC group. The cell viability, number of clones formed, scratch-healing rate, cell invasion, and expression levels of IMP3, PD-L1, CD44v6, MMP-2, and MMP-9 proteins were all significantly increased while expression of E-cadherin protein was significantly decreased in the miR-NC+IMP3 and miR-NC+PD-L1 groups compared with the miR-NC+pcDNANC group. The cell viability, number of clones formed, rate of scratch healing, cell invasion, and expression levels of IMP3, PD-L1, CD44v6, MMP-2, and MMP-9 protein were all significantly increased while E-cadherin expression was significantly decreased in the miR-34a-5p+IMP3 and miR-34a-5p+PD-L1 groups compared with the miR-34a-5p+pcDNANC group (P< 0. 05). Conclusions miR-34a-5p can inhibit the proliferation, migration, invasion, and epithelialmesenchymal transition of breast cancer cells by targeting the expression of IMP3 and PD-L1.