Several dietary agents, such as micronutrient and non-nutrient components, the so-called bioactive food components, have been shown to display anticancer properties and influence genetic processes. The most common epigenetic change is DNA methylation. Hypomethylation of long interspersed elements (LINE-1) has been associated with an increased risk of several cancers, although conflicting findings have also been observed. The aim of the present study was to test the hypothesis that a low adherence to the Mediterranean diet (MD) and folate deficiency may cause LINE-1 hypomethylation in blood leukocytes of healthy women, and thus genomic instability. One hundred and seventy-seven non-pregnant women were enrolled. Mediterranean diet score (MDS) and folate intake were calculated using a food frequency questionnaire. LINE-1 methylation level was measured by pyrosequencing analysis in three CpG sites of LINE-1 promoter. According to MDS, only 9.6 % of subjects achieved a high adherence to MD. Taking into account the use of supplements, there was a high prevalence of folate deficiency (73.4 %). Women whose consumption of fruit was below the median value (i.e., <201 gr/day) were 3.7 times more likely to display LINE-1 hypomethylation than women whose consumption was above the median value (OR 3.7; 95 % CI 1.4-9.5). Similarly, women with folate deficiency were 3.6 times more likely to display LINE-1 hypomethylation than women with no folate deficiency (OR 3.6; 95 % CI 1.1-12.1). A dietary pattern characterized by low fruit consumption and folate deficiency is associated with LINE-1 hypomethylation and with cancer risk.