<b><i>Background/Aims:</i></b> Investigation of the genetic makeup may facilitate the implementation of more personalized nutritional interventions. The aims were to examine whether the rs10830963 <i>MTNR1B</i> polymorphism affects weight loss in response to a hypocaloric diet and to find potential gene-gene interplays and gene-diet interactions. <b><i>Methods:</i></b> 167 subjects enrolled in a personalized nutritional intervention for weight loss (3-6 weeks) were examined for anthropometric measurements, dietary habits and physical activity at baseline and at the first follow-up visit. Three polymorphisms, which have previously been associated with body weight regulation, rs10830963 <i>(MTNR1B)</i>, rs9939609 <i>(FTO)</i> and rs17782313 <i>(MC4R)</i>, were analyzed using the Luminex® 100/200™ System. <b><i>Results:</i></b> After adjusting for covariates, females with the rs10830963 CG/GG genotype showed lower weight loss than those with the CC genotype. In the total population, carriers of variant alleles of both <i>FTO</i> and <i>MC4R</i> showed a significant association with <i>MTNR1B</i> and weight loss outcome. Moreover, among women, higher total protein and animal protein intakes were associated with a lower weight loss in G allele carriers of the <i>MTNR1B</i> variant. <b><i>Conclusions:</i></b> Our data evidenced that rs10830963 <i>MTNR1B</i> polymorphism could be associated with individual differences in weight loss induced by a hypocaloric diet. This association was influenced by <i>FTO</i> and <i>MC4R</i> loci and modified by baseline protein intake.