Objective To construct a microRNA (miRNA)-mRNA regulatory network in rats with calcium oxalate nephrolithiasis using bioinformatics technology, and to screen for the related core molecules regulating the formation of calcium oxalate nephrolithiasis. Methods The miRNA and mRNA datasets of rats with calcium oxalate nephrolithiasis induced by glycol feeding for 14 d were screened from the Gene Expression Omnibus (GEO) database. The differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) were analyzed by GEO2R online tool, and displayed by heatmap diagrams. The target genes of DEMs were predicted by miRWalker and then were intersected with DEGs to identify the regulatory genes by "venn.diagram" package of R software according to the principle of negative regulation of miRNA to mRNA. "Clusterprofile" package was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the regulatory genes, and the protein-protein interaction (PPI) analysis was performed by String database. Lastly, Cytoscape software was used to visualize the PPI and miRNA-mRNA regulatory network and screen the hub genes. Results A total of 38 DEMs and 364 DEGs were screened. Totally 116 regulatory genes were obtained through the intersection of DEMs' target genes and DEGs, and they were enriched in 24 GO items and 22 KEGG signaling pathways. The miRNA-mRNA regulatory network was successfully constructed, and the miRNAs at the core regulatory position were miRNA-6215, miRNA-758-5p, and miRNA-214-3p. Five hub genes were identified from PPI network, namely forkhead box M1 (Foxm1), nuclear division cycle 80 (Ndc80), cyclin dependent kinase inhibitor 2B (Cdkn2b), Cdc10-dependent transcript 1 (Cdt1), and inhibitor of nuclear factor κB kinase regulatory subunit gamma (Ikbkg). Conclusion Through constructing miRNA-mRNA regulatory network, miRNAs and genes that play key regulatory roles in the formation of calcium oxalate nephrolithiasis are obtained, which provides new research ideas for the prevention and treatment of calcium oxalate nephrolithiasis.