Background: MicroRNAs (miRNAs) play key roles in posttranscriptional regulation during the window of implantation. However, which miRNA may play regulatory role during the window of implantation remains to be studied in depth. This paper aimed to explore the miRNAs that played regulatory roles during the process of implantation. Methods: RNA sequencing was performed to analyze mice uterus tissue in gestation day 1 (D1), gestation day 4 (D4) and gestation day5 (D5). The tissues in D5 were divided into embryo implantation sites (D5IMS) and inter-implantation sites (D5IIS). The differentially expressed miRNAs were screened and bioinformatics analyzed. Transfecting miR-183-5p mimics into HEC-1-A cells, genes regulated by miR-183-5p were analyzed by transcriptome sequencing. Result: Eleven differentially expressed miRNAs were identified during the window of implantation. KEGG enrichment analysis showed that the most differentially expressed miRNAs mainly related to binding and signaling transduction related pathways. Especially miR-183-5p, miR-182-5p, miR-199b-5p and miR-218-5p play a crucial role in regulating many important pathways. Transcriptome sequencing results showed that there were 19 up-regulated and 31 down-regulated genes in the miR-183-5p mimics group compared with the negative control (NC) group. This work laid a foundation for the study of miRNA in early pregnancy.