Numb is an adaptor protein that controls the fate of cells in different species through asymmetrical inheritance by sibling cells during division. It has been investigated extensively in mitosis, mostly in neural progenitor cells, but its function in meiosis remains unknown. The present study was designed to investigate the expression, subcellular localisation and functional roles of Numb during mouse oocyte meiotic maturation. Using real-time polymerase chain reaction and western blotting, we found that the expression of Numb increased from the germinal vesicle (GV) to MII stages. Immunofluorescent staining revealed that Numb was mainly concentrated in the GV before meiosis resumption, aggregated in the vicinity of the chromosomes after GV breakdown and then localised to the spindle poles from prometaphase I to MII. Nocodazole treatment resulted in spindle destruction and Numb diffusion into the cytoplasm. However, Numb appeared at the spindle poles again once the spindles had formed when nocodazole-treated oocytes were washed and cultured for spindle recovery. Depletion of Numb by RNA interference resulted in chromosome misalignment, spindle deformation and even doubled spindle formation. Our results suggest that Numb is critical for spindle organisation during mouse oocytes meiosis. The present study provides evidence of a new function for Numb in addition to its action as a cell fate-determining factor.