Tributyltin oxide (TBTO) has been widely used as marine antifouling composition, preservative, biocide, and a stabilizer in plastic industry. Previous studies have indicated that TBTO can cause immunotoxicity as an environmental pollutant. However, little is known about its reproductive toxicity, especially on female oocyte maturation and the underlying mechanisms. In this study, mouse oocytes were cultured with different concentrations of TBTO in vitro, and several crucial events during meiotic maturation were evaluated. We found that the first polar body extrusion rate was significantly reduced, which reflected the disruption of meiotic maturation. The rate of abnormal spindle organization increased significantly, accompanied with a higher rate of chromosome misalignment. In addition, TBTO treatment increased reactive oxygen species generation markedly, which also accelerated the early-stage apoptosis. Moreover, heterogeneous mitochondrial distribution, mitochondrial dysfunction, and higher rate of aneuploidy were detected, which consequently disrupted in vitro fertilization. In conclusion, our results indicated that TBTO exposure could impair mouse oocyte maturation by affecting spindle organization, chromosome alignment, mitochondria functions, oxidative stress, and apoptosis.