Spindle checkpoint proteins control entry into anaphase and chromosome segregation. As a member of spindle checkpoint proteins, MAD2 takes a central role in the regulation of anaphase onset and genome integrity. Here, we used MAD2 siRNA transfection approach to study MAD2 functions during mouse oocyte meiotic maturation in vitro. Real-time PCR and laser scanning confocal microscopy showed that we successfully downregulated MAD2 transcript and protein expression. We further demonstrated that MAD2 downregulation resulted in a shortened duration of meiosis I and meiotic spindle abnormality, suggesting the function of MAD2 in mouse oocyte meiotic maturation. We also showed that MAD2 interference to some extent decreased GVBD rate, but increased apoptosis in mouse oocytes. In conclusion, our study shows that siRNA transfection is an effective tool to study MAD2 functions, and our results provide further evidence for the role of MAD2 as a spindle checkpoint protein in mouse oocytes.