Differentially methylated regions (DMR) are genomic regions with different methylation status. The aim of this research was to identify DMRs in subjects with obesity that predict the response to a weight-loss dietary intervention and its association with metabolic variables. Based on the change in body mass index (BMI), 201 subjects with overweight and obesity were categorized in tertiles according to their response to a hypocaloric diet: Responders (R; n = 64) and Non-Responders (NR; n = 63). The R group lost 4.55 +/- 0.91 BMI units (kg/m(2)) and the NR group lost 1.95 +/- 0.73 kg/m(2) (p < 0.001). DNA methylation was analysed in buffy coat through a methylation array at baseline. DMRs were analysed using a function of ChAMP (Chip Analysis Methylation Pipeline) in R software. Baseline DNA methylation analysis between R and NR exhibited a DMR located at paraoxonase 3 gene (PON3) consisting of 13 CpG sites, eleven of them significantly hypermethylated in R. To analyse the implication of these 11 CpGs on weight loss, a z-score was performed as a measure of DMR methylation. This analysis showed a correlation between PON3 DNA methylation and BMI loss. This z-score negatively correlated with PON3 protein serum levels. Total paraoxonase activity in serum was not different between groups, but PON enzymatic activity positively correlated with oxidized LDL levels. The present study identified a DMR within PON3 gene that is related to PON3 protein levels in serum, and that could be used as a potential biomarker to predict the response to weight-loss dietary interventions.