Abstract: Objective To explore the diagnostic value and clinical significance of miRNA-143 and miRNA-221 in older patients with rectal cancer. Methods Fifty older patients with rectal cancer were analyzed and 30 healthy individuals served as control from September 1, 2015 to December 30, 2016. We used high-throughput sequencing and fluorescence quantitative RT-PCR to detect and verify the miRNAs differential expression in the above samples. Pearson test was used to analyze the correlation between miRNA-143 and miRNA-221. The two miRNAs' target genes were predicted using the miRBase database. We used R 3.4.0 software to draw differential miRNA heat map clustering graphs. MiRNet database and cytoscape 3.5.1 were used to plot the miRNA and mRNA expression regulatory network. Results Compared with normal tissues, miRNA-143 and miRNA-221 were both down-regulated in elderly primary rectal cancer tissues (P<0.001, P=0.009), and their expression levels were positively correlated (r=0.517, P=0.003). GO functional annotation mainly enriched in biological process and molecular function, for instance, stem cell development, MAPKK activity, STAT protein tyrosine phosphorylation regulation, protein phosphorylation, tyrosine phosphorylation of amino acid binding peptide, regulating lipid kinase activity, polymerization, poly nuclear phosphoprotein mRNA A tail shortened (P<0.01). KEGG pathway analysis involved TP53 genes regulating cell death gene transcription, VEGF signaling pathway, IL-4 signaling pathway, oncostatin M signaling (P<0.01). The regulatory network indicates that there are a few target genes overlap between miRNA-143 and miRNA-221, and the two have synergistic effects. Conclusion As tumor suppressor factors of rectal cancer, miRNA-143 and miRNA-221 provide certain clinical significance for the diagnosis of senile rectal cancer.