Background:The fat mass-and obesity-Associated (FTO) gene influences energy homeostasis in humans. Although the obesity-related variant, rs9939609 has been replicated across a number of cohort studies, there remains significant variance and a low to modest association. Telomere length is another commonly reported obesity risk factor. We hypothesize understanding the associations between FTO rs9939609 with FTO methylation and telomere length will provide a more accurate assessment of obesity risk.Methods:Overall, 942 participants free of diabetes or pre-diabetes were included in the retrospective study. Leukocyte genomic DNA was analyzed for rs9939609 genotyping, FTO gene methylation and leukocyte telomere length (LTL) measurement.Results:In general linear models, rs9939609 AA genotypes were associated with increased fat percentage (3.15%, P=0.001), fat mass (4.16 kg, P=0.001), body mass index (BMI) (1.38, P=0.006) and waist circumference (3.35 cm, P=0.006), but not with FTO methylation or LTL in this overall population. However, when participants were stratified into higher and lower FTO methylation groups, the AA genotype possesses a 2.04-fold increased obesity risk in comparison to TT genotype (95%CI, 1.07-3.89, P=0.031) in participants with a higher FTO methylation level, but this association was absent in the lower FTO methylation sub-group. Moreover, AT and AA genotype carriers were associated with shorter LTL compared to TT carriers (P=0.020 and P=0.111, respectively) in the higher FTO methylation level group. However, this association was absent in the lower methylation group. Furthermore, FTO gene methylation level was significantly associated with LTL in the 942 samples (P=0.017).Conclusions:FTO rs9939609 is associated with obesity risk and LTL in this study, where this association is only observed at higher, but not lower, FTO methylation levels of participants. Our data suggest association of multiple factors, including FTO methylation level, may be involved in one of several mechanisms underlying the commonly reported obesity risk of this FTO polymorphism.