<b><i>Introduction:</i></b> Carbohydrate intake and physical activity are related to glucose homeostasis, both being influenced by individual genetic makeup. However, the interactions between these 2 factors, as affected by genetics, on glycaemia have been scarcely reported. <b><i>Objective:</i></b> We focused on analysing the interplay between carbohydrate intake and physical activity levels on blood glucose, taking into account a genetic risk score (GRS), based on SNPs related to glucose/energy metabolism. <b><i>Methods:</i></b> A total of 1,271 individuals from the Food4Me cohort, who completed the nutritional intervention, were evaluated at baseline. We collected dietary information by using an online-validated food frequency questionnaire, a questionnaire on physical activity, blood biochemistry by analysis of dried blood spots, and by analysis of selected SNPs. Fifteen out of 31 SNPs, with recognized participation in carbohydrate/energy metabolism, were included in the component analyses. The GRS included risk alleles involved in the control of glycaemia or energy-yielding processes. <b><i>Results:</i></b> Data concerning anthropometric, clinical, metabolic, dietary intake, physical activity, and genetics related to blood glucose levels showed expected trends in European individuals of comparable sex and age, being categorized by lifestyle, BMI, and energy/carbohydrate intakes, in this Food4Me population. Blood glucose was inversely associated with physical activity level (β = −0.041, <i>p</i> = 0.013) and positively correlated with the GRS values (β = 0.015, <i>p</i> = 0.047). Interestingly, an interaction affecting glycaemia, concerning physical activity level with carbohydrate intake, was found (β = −0.060, <i>p</i> = 0.033), which also significantly depended on the genetic background (GRS). <b><i>Conclusions:</i></b> The relationships of carbohydrate intake and physical activity are important in understanding glucose homeostasis, where a role for the genetic background should be ascribed.