Decidualization of endometrial stromal cells is an important feature of implantation and pregnancy. The molecular mechanism underlying decidualization remains unclear, particularly regarding the microRNA (miRNA/miR) regulation of this process. The present study revealed the temporal and spatial distribution of mmu-miR-96 in the mouse uterus during early pregnancy by reverse transcription-quantitative polymerase chain reaction and in situ hybridization. In addition, primary stromal cells were isolated from the mouse uterus and used to explore the role of mmu-miR-96 in decidualization. The results demonstrated that mmu-miR-96 was highly expressed in stromal cells during pregnancy, and was upregulated at implantation sites. In addition, mmu-miR-96 was strongly expressed during decidualization, which indicates that it may serve a role in the decidualization of stromal cells. Based on existing reports, mmu-miR-96 participates in apoptosis; therefore the present study investigated its effects on the apoptosis of primary endometrial stromal cells. The results indicated that overexpression of mmu-miR-96 may induce apoptosis of stromal cells. In further studies regarding the underlying mechanism, the target genes of mmu-miR-96 were screened by bioinformatics analysis, and it was confirmed that B-cell lymphoma 2, an anti-apoptotic gene, was the target of mmu-miR-96, as determined using a reporter gene assay. In conclusion, the present study suggested that mmu-miR-96 participates in the decidualization of endometrial stromal cells in mice, thereby serving a key role in pregnancy.