Preeclampsia (PE) is a life-threatening hypertensive renal disorder of pregnancy. Soluble fms-like tyrosine kinase 1 (sFlt-1), an anti-angiogenic factor, is elevated and contributes to PE. However, molecular basis underlying its up-regulation in PE remains unclear. Here we report that hypoxia inducible factor-1α (HIF-1α), an important transcriptional factor, and U2AF65, a key molecule involved in alternative splicing, are significantly increased in the placentasof PE patients compared to the controls. Using two animal models of PE including angiotensin receptor type 1 receptor agonistic autoantibody (AT1-AA) and LIGH (a TNF-α superfamily member)-induced PE mouse model coupled with in vivo nanoliposome-delivery system to specifically knockdown HIF-1α and U2AF65, we found that knockdown of HIF-1α or U2AF65 significantly attenuated AT1-AA-induced hypertension (HIF-1α siRNA: 137±4mmHg; U2AF65 siRNA: 140±5mmHg, vs. AT1-AA group 164±3mmHg, p<0.05), proteinuria (HIF-1α siRNA: 41.6±3.2μg/mg, 34.9±5.3μg/mg, vs. AT1-AA group 85.9±4.8μg albumin/mg creatinine, p<0.05) and circulating sFlt-1 level. Similarly, we found that HIF-1α and U2AF65 siRNA knockdown significantly reduced sFlt-1 levels, hypertension and proteinuria in LIGHT-infused pregnant mice. Finally, using splice-specific PCR assay, we found that knockdown HIF-1α significantly reduced total Flt-1 mRNA level in the placentas of both AT1-AA and LIGHT-infused pregnant mice but no effect on the ratio of sFlt-1/ Flt-1. In contrast, U2AF65 knockdown significantly reduced the ratio of sFlt-1/ Flt-1 without an effect on total Flt-1 mRNA level in these two PE mouse models. Similar to mouse, using human villous explant cultures (HVEx), we found that HIF-1α knockdown directly reduced total Flt-1 mRNA but not the ratio of sFlt-1/ Flt-1in AT1-AA or LIGHT-treated HVEx . While, U2AF65 siRNA knock down significantly reduced the ratio of sFlt-1/ Flt-1 but no effect on total Flt-1 mRNA levels in AT1-AA or LIGHT-treated HVEx. Overall, using both human and mouse studies, we have revealed two molecular basis underlying sFlt-1 inductions in PE: 1) elevated HIF-1α directly increases sFlt-1 levels under transcriptional levels; 2) elevated U2AF65 enhances alternative splicing to increase sFlt-1 levels.