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Background: Dietary macronutrients may indirectly affect body weight through their interactions with the fat mass and obesity associated (FTO) gene. This study aimed to investigate the association between FTO gene rs9939609 polymorphism with macronutrients intake in overweight adults. Methods: This study was carried out on 196 overweight adults of Shiraz, Iran. Dietary intake was assessed using a validated 168-item semi-quantitative food frequency questionnaire (FFQ). The FTO gene was genotyped for rs9939609 polymorphism. The association between dietary macronutrients and the FTO genotype were assessed using linear regression after adjustments for sex, age, physical activity, and the serum levels of triglycerides, fasting blood sugar (FBS), and low density lipoprotein (LDL). Results: The higher intake of carbohydrates (P < 0.001), fat (P = 0.009), and calorie (P = 0.001) were significantly associated with rs9939609 AA genotype (P = 0.001). Carriers of the AA genotype of rs9939609 had significantly higher calorie, fat, and carbohydrate intake than the carriers of the TT genotype after adjusting for age and sex (P = 0.019, P = 0.010 and P = 0.001, respectively). Further adjustments for physical activity, TG, LDL, and FBS did not change these results. Conclusion: The amounts of dietary calorie, carbohydrate, and fat intake were associated with FTO genotype. Further studies are warranted to confirm these associations and to identify the underlying mechanisms. [ABSTRACT FROM AUTHOR]