Gonadotoxic chemotherapeutics, such as cyclophosphamide, can cause early menopause and infertility in women. Earlier histological studies showed ovarian reserve depletion via severe DNA damage and apoptosis, but others suggested activation of PI3K/PTEN/Akt pathway and follicle 'burn-out' as a cause. Using a human ovarian xenograft model, we performed single-cell RNA-sequencing on laser-captured individual primordial follicle oocytes 12 h after a single cyclophosphamide injection to determine the mechanisms of acute follicle loss after gonadotoxic chemotherapy. RNA-sequencing showed 190 differentially expressed genes between the cyclophosphamide- and vehicle-exposed oocytes. Ingenuity Pathway Analysis predicted a significant decrease in the expression of anti-apoptotic pro-Akt PECAM1 (p = 2.13E-09), IKBKE (p = 0.0001), and ANGPT1 (p = 0.003), and reduced activation of PI3K/PTEN/Akt after cyclophosphamide. The qRT-PCR and immunostaining confirmed that in primordial follicle oocytes, cyclophosphamide did not change the expressions of Akt (p = 0.9), rpS6 (p = 0.3), Foxo3a (p = 0.12) and anti-apoptotic Bcl2 (p = 0.17), nor affect their phosphorylation status. There was significantly increased DNA damage by γH2AX (p = 0.0002) and apoptosis by active-caspase-3 (p = 0.0001) staining in the primordial follicles and no change in the growing follicles 12 h after chemotherapy. These data support that the mechanism of acute follicle loss by cyclophosphamide is via apoptosis, rather than growth activation of primordial follicle oocytes in the human ovary.