Preeclampsia (PE) is a pregnancy disease characterized by insufficient invasion and growth of trophoblast cells. adeno-associated virus encoding alkB homolog 1 (ALKBH1) is a demethylase in 5-methylcytosine (m5C) methylation modification. This study was performed to explore the role of ALKBH1 in hypoxia treated human extravasated trophoblast cells. Hypoxia treated human extravasated trophoblast cells (HTR-8/SVneo) was used to simulate the occurrence of PE in vitro. The cells phenotype was detected by CCK-8 and Transwell assays. The m5c levels and m5C levels of PSMD14 were analyzed by m5C dot blot and M5C Me-RIP assays. Then, the interaction between ALKBH1 and PSMD14 were confirmed by RIP and dual-luciferase reporter assays. ALKBH1 was up-regulated in hypoxia treated HTR-8/SVneo cells. Additionally, ALKBH1 knockdown increased the m5C contents, cell viability, migration and invasion abilities of hypoxia treated HTR-8/SVneo cells. Furthermore, ALKBH1 knockdown increased the m5C and mRNA levels, and mRNA stability of PSMD14. RIP and dual-luciferase reporter assays demonstrated that ALKBH1 interacted with PSMD14. Besides, PSMD14 knockdown reversed the effects of ALKBH1 silencing on cell viability, migration and invasion abilities of hypoxia treated HTR-8/SVneo cells. ALKBH1 mediated m5C levels were decreased in the hypoxia treated HTR-8/SVneo cells, which further decreased the cell viability, migration and invasion abilities through targeting the PSMD14 levels.