Meg8-DMR is the first maternal methylated DMR to be discovered in the imprintedDlk1-Dio3domain. The deletion ofMeg8-DMR enhances the migration and invasion of MLTC-1 depending on the CTCF binding sites. However, the biological function ofMeg8-DMR during mouse development remains unknown. In this study, a CRISPR/Cas9 system was used to generate 434 bp genomic deletions ofMeg8-DMR in mice. High-throughput and bioinformatics profiling revealed thatMeg8-DMR is involved in the regulation of microRNA: when the deletion was inherited from the mother (Mat-KO), the expression of microRNA was unchanged. However, when the deletion occurred from the father (Pat-KO) and homozygous (Homo-KO), the expression was upregulated. Then, differentially expressed microRNAs (DEGs) were identified between WT with Pat-KO, Mat-KO, and Homo-KO, respectively. Subsequently, these DEGs were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) term enrichment analysis to explore the functional roles of these genes. In total, 502, 128, and 165 DEGs were determined. GO analysis showed that these DEGs were mainly enriched in axonogenesis in Pat-KO and Home-KO, while forebrain development was enriched in Mat-KO. Finally, the methylation levels of IG-DMR,Gtl2-DMR, andMeg8-DMR, and the imprinting status ofDlk1,Gtl2,andRianwere not affected. These findings suggest thatMeg8-DMR, as a secondary regulatory region, could regulate the expression of microRNAs while not affecting the normal embryonic development of mice.