Down syndrome (DS) is one of the most common chromosomal abnormalities associated with congenital heart defects (CHD), with approximately 40 to 60% of cases showing cardiac defects. This study assessed (i) the association between maternal LINE-1 methylation and the occurrence of CHDs in children with DS and (ii) the impact of endogenous maternal factors ( C677T polymorphism and maternal age) and exogenous maternal factors (cigarette smoking, alcohol intake, medication use, body mass index and dietary habits such as folate intake) on maternal LINE-1 methylation and on the occurrence of CHD in children with DS. The study included 90 mothers of children with DS of maternal origin (49% DS-CHD mothers/51% DS-CHD mothers). LINE-1 DNA methylation was analyzed in peripheral blood lymphocytes by quantification of LINE-1 methylation using the MethyLight method. C677T polymorphism genotyping was performed using PCR-RFLP. LINE-1 methylation was not significantly different between DS-CHD and DS-CHD mothers ( = 0.997). Combination of C677T genotype/diet and BMI were significant independent predictors of LINE-1 DNA methylation in DS-CHD mothers (β -0.40, = 0.01 and β -0.32, = 0.03, respectively). In the analyzed multivariate model (model = 0.028), these two factors explained around 72% of the variance in LINE-1 DNA methylation in mothers of children with DS and CHD. The group with the highest BMI (≥30 kg/m2) had significantly lower LINE-1 methylation than the group with normal BMI (Bonferroni = 0.03) and the overweight group (Bonferroni = 0.04). The lowest LINE-1 DNA methylation values were found in DS-CHD mothers with the CT+TT genotype and a low-folate diet; the values were significantly lower than the values in mothers with the CC genotype and a folate-rich diet (Bonferroni = 0.04). Association between maternal LINE-1 methylation and CHD in children with DS was not found. Study showed that the genotype/diet combination and BMI were significantly associated with LINE-1 methylation in mothers of children with DS-CHD . These results highlight the need for a multifactorial approach to assess the roles of endogenous and exogenous maternal factors in maternal LINE-1 DNA methylation and the consequent pathologies in children. More extensive studies in a larger sample may help elucidate these relationships.