Aim The effect of miR-223-3p on H9c2 cells in high glucose environments was investigated through bioinformatics and its role in the mechanism of development of diabetic cardiomyopathy was analyzed in conjunction with transcriptomic sequencing results. The objective was to identify novel therapeutic targets at the molecular level and explore the specific mechanisms of action of miR-223-3p. Methods In high glucose-cultivated H9c2 cells, miR-223-3p inhibition and control were transfected, respectively. RT-qPCR was used to detect the differences in miR-222-3p expression between the two cell groups. Differential mRNA was identified through high-throughput sequencing. GO functional analysis was conducted using TopGO software. DESeq2 software (v1.16.1) filtered differentially expressed genes and analyzed them using a miR-223-3p target gene database. This process predicted the target genes of miR-223-3p and validated the changes in their expression through RT-qPCR. Results The activity of H9c2 cells treated with high glucose decreased significantly. Significant differences in gene expression between the control group and the inhibitor group had been indicated by transcriptomic sequencing results. GO function enrichment analysis showed that the predicted target gene set was significantly enriched in G protein-coupled receptor activity, glycerol ether monooxygenase activity, cellular anion homeostasis, and chloride ion homeostasis, among others. KEGG pathway enrichment analysis further showed that these genes were mainly involved in the TNF signaling pathway and the IL-17 signaling pathway. In addition, they were related to type 1 diabetes, cytochrome P450 metabolism of exogenous drugs, and other diseases and physiological processes. Target gene prediction suggested that miR-223-3p may be associated with the expression changes of Cxcl10, Creb3l3, Mmp3, and Bcl3, among others. Conclusion The prediction of miR-223-3p and its downstream target genes in high glucose induced H9c2 cell injury may provide new targets for the treatment of diabetic cardiomyopathy, which is of great significance for revealing the pathogenesis of diabetic cardiomyopathy and developing new treatment strategies.