Abstract: The aim of this study was to predict candidate genes for bovine microRNA-223 (bta-miR-223), and to perform protein interaction analysis and signal pathway enrichment analysis to construct a regulatory network in which it may participate. This study was conducted to investigate the relationship between mastitis resistance and bta-miR-223 by predicting the candidate target genes and analyzing the associated protein interaction, signal pathway enrichment and the regulatory networks. Targetscan and miRWalk were used to predict its candidate target genes. Protein association analysis and KEGG pathway analysis were performed using STRING, and DAVID, respectively. Cytoscape software was used to visualize the regulatory network in which the bta-miR-223 candidate target genes may be involved. The results indicated that there exist protein interactions among candidate target genes, for example FBXO30 and SMURF2, FBXW7 and UBA2, etc. In addition, the reactome pathways they participate in include antigen processing, the immune system, the innate immune system, and neutrophil degranulation. The results of KEGG pathway enrichment analysis showed that these candidate target genes are involved in epithelial cell bacterial invasion, endocytosis, PI3K-Akt, TGF-β, AMPK and other signaling pathways. The candidate target genes of bta-miR-223, FBXO30, SMURF2, FBXW7, UBA2, etc., may participate in antigen processing, innate immune system and neutrophil degranulation. Moreover, they may play an important regulatory role through bacterial invasion of epithelial cells, endocytosis, and PI3K-Akt signaling pathway.