Abstract: Meiosis is essential for oocyte maturation and embryonic development, representing a critical factor in mammalian reproduction. Consequently, assessing the impact of space microgravity on this process is paramount for evaluating reproductive health during long-term space missions. This article used a Random Positioning Machine (RPM) to simulate microgravity effects, examining how random changes in orientation relative to the gravity vector affect mouse oocyte meiosis. This study aims to provide critical biological references for further investigation into the damage mechanisms of space microgravity environments on oocyte meiotic division. Germinal Vesicle (GV)-stage mouse oocytes were encapsulated in Polydimethylsiloxane (PDMS) chip chambers to ensure stable culture conditions and precise positional control during RPM operation. Oocytes were cultured under RPM (RPM group) and static Normal Gravity (NG group) conditions. Meiotic progression was tracked and quantitatively analyzed at five key developmental stages: GV (0 h), GV Breakdown (GVBD, 2 h), Pro-Metaphase I (Pro-MI, 5 h), Metaphase I (MI, 8 h), and Metaphase II (MII, 16 h). Mitochondrial distribution, spindle morphology, and chromosome alignment were quantified through confocal laser microscopy coupled with fluorescent probes. The results showed that RPM condition reduced oocyte maturation rates by 32.75% compared to Normal Gravity (NG) controls (p<0.01). Mitochondrial dynamics exhibited stage-specific perturbations: perinuclear clustering at MI-stage (70.00% vs. 41.18% in NG) and disorganized aggregation patterns in 71.88% of MII-stage oocytes. In addition, spindle assembly and chromosome alignment were also disrupted: multipolar spindles during MI-stage caused disordered chromosome arrangement. At MII, RPM group oocytes displayed exacerbated spindle defects (57.58% abnormality rate vs. 22.32% in NG, p<0.05) and widened equatorial plates (15.63 μm vs. 7.55 μm, p<0.0001). These findings suggest that the simulated microgravity effect leads to abnormal meiosis and the decline of oocyte quality through tripartite disruption of mitochondrial-spindle-chromosomal coordination. This study significantly contributes to understanding how gravity changes affect oocyte meiosis.