Abnormal regulation of placental apoptosis and proliferation has been implicated in placental disorders. Recently, several DNA-damaging agents were reported to induce excessive apoptosis and reduce cell proliferation in the placenta; however, the molecular pathways of these toxic effects on the placenta are unclear. The aim of the present study was to determine the involvement of TRP53, a tumor suppressor that mediates cellular responses to DNA damage, in the induction of apoptosis and cell cycle arrest in the developing placenta. For this purpose, we treated pregnant mice on Day 12 of gestation with 10 mg/kg of etoposide and 5-Gy gamma irradiation, potent inducers of DNA damage. We found an increase in the number of trophoblastic apoptoses 8 and 24 h after etoposide injection and 6 and 24 h after irradiation in the placental labyrinth zone. The number of mitoses and DNA syntheses in trophoblasts decreased after treatment. The accumulation and phosphorylation of TRP53 protein were detected 8 and 6 h after etoposide injection and irradiation, respectively. In Trp53-deficient placentas, the induction of etoposide-induced trophoblastic apoptosis is abrogated, while the reduction of proliferation occurred similarly as in wild-type placentas. CDC2A, a regulator of G2/M progression, was inactivated by phosphorylation after etoposide injection and irradiation, suggesting that the cell cycle was arrested at the G2/M border by treatment. Our study demonstrated that etoposide injection induced TRP53-dependent apoptosis and TRP53-independent cell cycle arrest in labyrinthine trophoblasts, providing insights into the molecular pathway of placental disorders.