Preeclampsia (PE) is a pregnancy-specific disorder representing a major cause for maternal and perinatal morbidity and mortality. The dysfunction of trophoblast cells plays an important role in the pathogenesis of PE. In recent years, microRNAs (miRNAs) have been suggested to play an important role in regulating trophoblast cell biological functions involved in the pathogenesis of PE. Accumulating evidence has showed that miR-454 plays an important role in regulating cell functions. However, whether miR-454 is involved in regulating cell functions of trophoblast cells during PE remains unclear. In this study, we found that miR-454 expression was significantly downregulated in placental tissues from PE patients. in vitro experiments showed that miR-454 overexpression significantly increased proliferation, inhibited apoptosis, and promoted invasion of trophoblast cells, whereas miR-454 inhibition markedly suppressed proliferation, increased apoptosis, and inhibited invasion of trophoblast cells. Interestingly, bioinformatics analysis predicted that ephrin receptor B4 (EPHB4), an important gene for regulating trophoblast cell function in PE, was a potential target gene of miR-454. Dual-luciferase reporter assay showed that miR-454 directly targeted the 3’-untranslated region of EPHB4. Real-time quantitative polymerase chain reaction and Western blot analysis demonstrated that miR-454 negatively regulated EPHB4 expression in trophoblast cells. Moreover, miR-454 expression was found inversely correlated with EPHB4 expression in placental tissues from PE patients. Importantly, EPHB4 overexpression partially reversed the promotion effect of miR-454 overexpression on trophoblast cell proliferation and invasion. Taken together, these findings demonstrate that miR-454 promotes the proliferation and invasion of trophoblast cells by inhibiting EPHB4 expression, and the decreased miR-454 expression may contribute to PE by promoting EPHB4 expression. Our study provides novel insights into understanding the molecular pathogenesis of PE.