Background: Voriconazoleis(VRC) often used in complex therapeutic environments for the treatment and prevention of invasive fungal infections. The steady-state valley concentration (Cminss) of VRC not only varies between individuals, but also within individuals, which is difficult to fully explain by pharmacogenomic theory. It is necessary to propose a new perspective to explain the variation of voriconazole steady-state valley concentration. Objectives: Based on the regulation of ADME gene expression by DNA methylation, this study aimed to explore the effect of CYP2C19 DNA methylation level on the VRC Cminss. Methods: In this study, 116 concentration points were divided into low concentration group (Cminss<1.0mg/L), standard concentration group (Cminss =1.0-5.5mg/L) and high concentration group (Cminss>5.5mg/L) according to Voriconazole Cmin standard range of 1.0-5.5 mg/L. The effect of CYP2C19 DNA methylation was highlighted by predisposition score matching to exclude other confounding factors. Results: The CYP2C19 CpG25 methylation level was different between low concentration group and standard concentration group (p=0.047). There was no difference in the CYP2C19 DNA methylation between the high concentration group and the standard concentration group, but there were significant differences in CRP (p<0.001), Alb (p=0.007) and T-BIL (p=0.024) between the high concentration group and the standard concentration group. Conclusions: The VRC Cminss in the low concentration group may be related to the methylation degree of CYP2C19 CpG25 site, while the VRC Cminss in the high concentration group may be unrelated to the methylation degree of CYP2C19 but related to the levels of CRP, Alb and T-BIL.