Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1) downregulates insulin signaling by inhibiting the insulin receptor's tyrosine-kinase. K121Q and other ENPP1 single-nucleotide polymorphisms (SNPs), IVS20delT-11 and A/G+1044TGA, have been previously associated with obesity in French children, and the risk haplotype QdelTG has also been associated with this condition in both French and German children. Our aim was to perform a case-control replication study in order to assess the possible association of childhood obesity and overweight with the above-mentioned ENPP1 SNPs, and with the QdelTG haplotype, in the Italian population. A total of 865 healthy Italian children were studied: 453 normal-weight, 243 overweight and 169 obese subjects. Genotyping was performed by Taq-Man or Light-Cycler Technology. The Q variant of K121Q showed a negative association with overweight-obesity under both additive (odds ratio (OR) = 0.74, 95% confidence interval (CI) = 0.57-0.97, P = 0.030) and recessive (OR = 0.32, 95% CI = 0.10-0.97, P = 0.035) modes of inheritance. The Z-score of BMI showed a significant decreasing trend from children K/K homozygous to K/Q heterozygous, and to Q/Q homozygous (0.45 vs. 0.28 vs. -0.19; P = 0.009), according to the additive model. The two other SNPs and the QdelTG haplotype did not exhibit any association with overweight/obesity. This is the first child-based study showing a protective role of the 121Q variant of ENPP1 against overweight/obesity.