Calcium oxalate stones account for >80% of urinary stones, however the mechanisms underlying their formation remains to be elucidated. Hyperoxaluria serves an important role in the pathophysiological process of stone formation. In the present study, differences in the miRNA expression profiles between experimental hyperoxaluric rats and normal rats were analyzed, in order to identify target genes and signaling pathways involved in the pathogenesis of hyperoxaluria. Ethylene glycol and ammonium chloride was fed to male hyperoxaluric rats (EXP) and normal age-matched male rats (CON). The oxalate concentration in the urine of each experimental rat was collected every 24 h and measured on day 14. Three rats exhibiting the highest concentrations were selected for microarray analysis. Microarray analysis was performed to evaluate differences in the expression of microRNA (miRNA) in the kidney tissues from EXP and CON groups, and miRNAs that exhibited a > 2-fold or a <0.5-fold alteration in expression between these groups were screened for differential expression patterns according to the threshold P-values. Reverse transcription-quantitative polymerase chain reaction analysis was employed to confirm the microarray results. In order to predict the potential role of miRNAs in pathophysiological processes, gene ontology (GO), pathway and target prediction analyses were conducted. A total of 28 miRNAs were observed to be differentially expressed (> 2-fold change) between EXP and CON groups. Among these miRNAs, 20 were upregulated and 8 were downregulated. GO and pathway analyses revealed that the insulin resistance and phosphatidylinositol-bisphosphonate 3-kinase/ AKT serine threonine kinase signaling pathways were potentially associated with miRNA regulation in this setting. In conclusion, the results of the present study identified differentially expressed miRNAs in hyperoxaluric rats, and provided a novel perspective for the role of miRNAs in the formation of calcium oxalate stones.