Genetic susceptibility may influence ischemic heart disease (IHD) predisposition and affect coronary blood flow (CBF) regulation mechanisms. The aim of this study was to investigate the association among single nucleotide polymorphisms (SNPs) of genes encoding for proteins involved in CBF regulation and IHD. A total of 468 consecutive patients were enrolled and divided into three groups according to coronary angiography and intracoronary functional tests results: G1, patients with coronary artery disease (CAD); G2, patients with coronary microvascular dysfunction (CMD); and G3, patients with angiographic and functionally normal coronary arteries. A genetic analysis of the SNPs rs5215 of the potassium inwardly rectifying channel subfamily J member 11 (KCNJ11) gene and rs1799983 of the nitric oxide synthase 3 (NOS3) gene, respectively encoding for the Kir6.2 subunit of ATP sensitive potassium (KATP) channels and nitric oxide synthase (eNOS), was performed on peripheral whole blood samples. A significant association of rs5215_G/G of KCNJ11 and rs1799983_T/T of NOS3 genes was detected in healthy controls compared with CAD and CMD patients. Based on univariable and multivariable analyses, the co-presence of rs5215_G/G of KCNJ11 and rs1799983_T/T of NOS3 may represent an independent protective factor against IHD, regardless of cardiovascular risk factors. This study supports the hypothesis that SNP association may influence the crosstalk between eNOS and the KATPchannel that provides a potential protective effect against IHD.