This experiment investigated the optimal time required for cryopreserved human oocytes to reform their spindles upon re-warming. Metaphase II oocytes were cryopreserved with a slow freezing method. Oocytes from each patient were randomly allocated into one of the 3 groups with different culture periods: 1, 3, or 5 hours, respectively, after thawing. Tubulin and chromosome configurations were visualized by confocal microscopy after immunostainings. By morphological assessment, 87.3% oocytes survived the process of freezing and thawing. Oocytes with normal spindle configuration increased significantly after 3 or 5 hours of incubation compared to those incubated for only 1 hour ( P < 0.05). There were no differences in the chromosome configurations among the treatment groups ( P > 0.05). This experiment demonstrated that cryopreserved human oocytes need a certain minimum period of incubation time (3 h) to recover their disrupted MII spindles and this information can be used in development of human IVF protocols with frozen oocytes.