Although in vitro maturation (IVM) of oocytes has been used for a relatively long time, during which the culture conditions have improved remarkably, the resulting germ cells are still not fully comparable to the cells obtained from the ovary in many important aspects, namely in fertilization rate and subsequent embryonic development. Some of the differences between IVM and in vivo maturation (IVV) oocytes were already discovered, including variability in spindle assembly and morphology. In this study we focused on a role of molecular motor Kif11 (hereafter referred to as Eg5) in maintaining bipolar spindle structure in IVM and IVV oocytes. Our experiments revealed that in IVM oocytes, Eg5 is abundant on meiosis II spindle, which makes these cells more sensitive to Eg5 inhibition than IVV oocytes. We further demonstrate that this sensitivity is acquired gradually with exposure to the in vitro conditions. This is a remarkable difference in function of spindle apparatus between IVM and IVV oocytes, and we believe our results are important not only for understanding of the chromosome segregation in mammalian oocytes but also because they indicate cells are using alternative pathways to achieve the same function when exposed to different conditions.