The objective of this study was to elucidate the dynamics of microtubules in post-ovulatory aging in vivo and in vitro of mouse oocytes. The fresh ovulated oocytes were obtained from oviducts of superovulated female ICR mice at 16 hours after hCG injection. The post-ovulatory aged oocytes were collected at 24 and 48 hours after hCG injection from in vivo and in vitro, respectively. Immunocytochemistry was performed on ${eta}$-tubulin and acetylated ${alpha}$-tubulin. The microtubules were localized in the spindle assembly, which was barrel-shaped or slightly pointed at its poles and located peripherally in the fresh ovulated oocytes. The frequency of misaligned metaphase chromosomes were significantly increased in post-ovulatory aged oocytes after 48 hours of hCG injection. The spindle length and width of post-ovulatory aged oocytes were significantly different from those of fresh ovulated oocytes, respectively. The staining intensity of acetylated ${alpha}$-tubulin showed stronger in post-ovulatory aged oocytes than that in the fresh ovulated oocytes. In the aged oocytes, the spindles had moved towards the center of the oocytes from their original peripheral position and elongated, compared with the fresh ovulated oocytes. Microtubule organizing centers were formed and observed in the cytoplasm of the aged oocytes. On the contrary, it was not observed in the fresh ovulated oocytes. The alteration of spindle formation and chromosomes alignment substantiates the poor development and the increase of disorders from the post-ovulatory aged oocytes. It might be important to fertilize on time in ovulated oocytes for the developmental competence of embryos with normal karyotypes.