Purpose MicroRNA-21 (miR-21) is a well-known oncomiR and plays key roles in regulating various biological processes related to pulmonary diseases, especially lung carcinoma. The regulatory roles and downstream targets of miR-21 remain far from well understood. We aimed to identify miR-21-gene regulatory network in lung tissue. Methods Transcriptome and proteome analyses were performed on lung tissues from miR-21 knockout (KO) mice and their wildtype (WT) littermates. Differentially expressed genes (DEGs) and proteins (DEPs) between miR-21KO and WT were analyzed, and correlation analysis was performed between transcriptional and translational level. DEPs were used for prediction of miR-21 target genes and construction of co-expression network. Results Comparing with WT mice, 820 DEGs and 623 DEPs were identified in lung tissues of miR-21KO mice. Upregulated DEGs and DEPs were both significantly enriched in pathways of metabolism of xenobiotics by cytochrome P450, drug metabolism, and chemical carcinogenesis. Of the 31 molecules commonly identified in DEGs and DEPs, 9 upregulated genes were tumor suppressor genes while 8 downregulated genes were oncogenes, and 12 genes showed closely positive correlation between mRNA and protein expression. Real-time PCR validation results were consistent with the omics data. Among the upregulated DEPs in miR-21KO mice, 21 genes were predicted as miR-21 targets. The miR-21 regulatory network was constructed by target genes and their highly co-expressed proteins, which identified the miR-21 target Itih4 as a hub gene. Conclusion MiR-21-gene regulatory network was constructed in mouse lung tissue. MiR-21KO resulted in extensive upregulation of tumor suppressor genes and downregulation of oncogenes.