Using an integrative approach where genetic variation, gene expression and clinical phenotypes are assessed in relevant tissues may help functionally characterize the genetic contribution to disease susceptibility. We sought to identify genetic variation influencing skeletal muscle gene expression (eQTL), as well as expression associated with measures of insulin sensitivity. We investigated associations of 3799401 genetic variants with gene expression from >7000 genes from three cohorts (n=104). We identified 287 genes with cis-acting eQTLs (FDR<5%; P<1.96x10(-5)) and 49 expression-insulin sensitivity phenotype associations (i.e. fasting insulin, HOMA-IR and BMI) (FDR<5%; P=1.34x10(-4)). One of these associations, fasting insulin/phosphofructokinase (PFKM), overlaps with an eQTL. Further, the expression of PFKM, a rate-limiting enzyme in glycolysis, was nominally associated with glucose uptake in skeletal muscle (P=0.026, n=42) and over-expressed (PBF-corrected=0.03) in skeletal muscle in T2D patients (n=102) compared with normoglycemic controls (n=87). The PFKM eQTL (rs4547172; PeQTL=7.69x10(-6)) was nominally associated with glucose uptake, glucose oxidation rate, intramuscular triglyceride content, and metabolic flexibility (P=0.016-0.048, n=178). We explored eQTL results using published GWAS data (from DIAGRAM and MAGIC) and a proxy for the PFKM eQTL (rs11168327, r2=0.75), was nominally associated with T2D (PDIAGRAM=2.7x10(-3)). Taken together, our analysis highlights PFKM as a potential regulator of skeletal muscle insulin sensitivity.