The impact of folate deficiency on global DNA methylation is uncertain. It also is unclear whether global DNA methylation is associated with outcome in HCC. LINE-1 methylation levels, as a surrogate marker of global methylation, may be influenced by folate deficiency. However, the interaction between LINE-1 methylation and folate level on overall survival (OS) in hepatocellular carcinoma (HCC) patients is unknown. We evaluated whether LINE-1 hypomethylation and folate deficiency are associated with HCC prognosis. We prospectively recruited 172 HCC patients between 2008 and 2012. LINE-1 methylation levels in plasma and white blood cells (WBC) were measured by pyrosequencing, and plasma folate levels by a radioprotein-binding assay. Patients with plasma LINE-1 methylation <70.0% (hypomethylation) had significantly worse OS compared with those with ≥70.0% methylation (hypermethylation) [hazard ratio (HR)=1.77; 95% confidence interval (CI) 1.12-2.79; P=0.015]. HCC patients with lower plasma folate levels also had worse survival (<27.7 vs. ≥27.7nmol/L; HR=1.96; 95% CI, 1.24-3.09; P=0.004). Furthermore, survival was poor in patients in whom both plasma LINE-1 methylation and folate levels were low compared with those patients in whom both levels were high (HR=3.36; 95%CI, 1.77-6.40; P<0.001). This interaction neared statistical significance (P=0.057). No significant association was found between WBC LINE-1 methylation levels and survival. These findings suggest that both lower plasma levels of LINE-1 methylation and folate are associated with worse survival in HCC patients.