Abnormal growth and migration of trophoblast cells is one of the main causes of spontaneous abortion. Eukaryotic translation initiation factor 5A (eIF5A) plays an important role in trophoblast cell growth and migration; however, its underlying mechanism remains largely unknown. Here, we first confirmed that eIF5A knockdown reduced human chorionic trophoblast HTR8 cells viability, proliferation, and migration. Next, we sought to systematically identify the genes regulated by eIF5A and observed changes in the transcriptome profile of eIF5A-knockdown HTR8 cells by RNA-seq analysis. Especially, we found that inhibition of eIF5A reduced both the mRNA and protein levels of methyltransferase-like protein 14 (METTL14). Furthermore, inhibition of METTL14 expression resulted in the reduction of viability, proliferation, and migration of HTR8 cells. In addition, we showed that overexpression of METTL14 rescued the effects of eIF5A knockdown in HTR8 cells. Finally, we revealed that eIF5A and METTL14 expression was decreased in spontaneous abortion samples compared to that in elective-induced abortion samples. Collectively, our study demonstrated that eIF5A plays a crucial role in HTR8 cells via modulation of METTL14 expression and may serve as a novel potential target for spontaneous abortion diagnosis and treatment.