In ascidian eggs, cytoplasmic and cortical reorganization, previously called ooplasmic segregation, occurs in two phases during the first cell cycle. In the second phase of reorganization, the mitochondria-rich cytoplasm (myoplasm) moves to the future posterior side, concurrent with sperm aster migration along the egg cortex. Although this reorganization is the critical step for establishing the anteroposterior axis, its molecular mechanism is not fully understood. In this study, we showed that low concentrations of the mitochondrial inhibitor sodium azide (NaN3 ), which showed the low toxicity in sperm, inhibited the second phase of reorganization without the microtubule depolymerization. In the NaN3 -treated embryo, the sperm aster was not attracted to the cortex and altered its migration pathway; therefore, the myoplasm remained at the vegetal pole. Consequently, the anteroposterior axis was not established. Another mitochondrial inhibitor, oligomycin, did not affect these processes. These results suggest that NaN3 inhibits unknown molecules that are important for the second phase of reorganization. Identifying the target molecule of NaN3 will lead to a molecular understanding of cytoplasmic and cortical reorganization.