Preeclampsia (PE) is a severe pregnancy-induced disorder characterized by hypertension and proteinuria and a leading cause of perinatal maternal-fetal mortality and morbidity in developing countries. Dysregulated human leukocyte antigen (HLA)-G was found in placentas as well as in maternal sera from PE patients; however, the reason for this difference is unknown. As accumulating evidence has confirmed that DNA methylation is an important mechanism for regulating gene expression, we sought to test the hypothesis that alteration in the DNA methylation of the HLA-G promoter region is responsible for decreased expression of HLA-G in PE. Bisulfite pyrosequencing showed that a series of CpG sites in the HLA-G promoter region were significantly more highly methylated in PE than in normal pregnancy (NP). Interestingly, the hypermethylated CpG sites were mostly reported to be binding sites of active transcription factors. To further investigate the regulation of HLA-G methylation, we also defined the expression patterns of DNA methyltransferases (DNMTs) in placental tissue using immunohistochemistry and quantitative polymerase chain reaction analyses. Here, we demonstrate that DNMT-1 is overexpressed and HLA-G expression is reduced in PE women when compared with NP. Furthermore, both treatment with the DNMT inhibitor 5-aza-2'-deoxycytidine and specific knockdown of DNMT-1 using siRNAs can significantly increase the expression level of HLA-G in a trophoblastic cell line, indicating the potential mechanism of DNMT-1-mediated DNA methylation in HLA-G regulation. Taken together, our research confirms that DNMT-1-mediated promoter hypermethylation of HLA-G is associated with PE. These findings provide new insights into the diagnosis and treatment of PE.