Type 2 diabetes (T2D) and obesity are strongly associated with low natriuretic peptide (NP) plasma levels and impaired NP signaling with a down-regulation of NP guanylyl cyclase receptor-A (GCA) in skeletal muscle and adipose tissue. However, no study has so far provided evidence for a causal link between ANP/GCA deficiency and T2D development. Here, we show that both ANP and GCA deficiency in mice promotes metabolic disturbances and prediabetes. In GCA haploinsufficient mice, skeletal muscle insulin resistance is associated with altered mitochondrial function and impaired endurance running capacity. Muscle RNA-seq analyses reveal down-regulation of various gene sets related to mitochondrial protein complexes and translation. Despite normal mitochondrial content, GCA haploinsufficient mice exhibit increased proton leak and reduced content of mitochondrial oxidative phosphorylation (OXPHOS) proteins. Using various clinical studies and cohorts, we further show that GCA is related to various metabolic traits in T2D and positively correlates with markers of oxidative capacity in human skeletal muscle. Altogether, these results indicate that ANP/GCA signaling controls muscle mitochondrial integrity and oxidative capacity in vivo and plays a causal role in the development of T2D. Overall design: Twelve weeks old Nppa-/- and Npr1+/- male mice (B6.129P2-Nppatm1Unc/J and B6.129-Npr1tm1Gar/J mice were backcrossed to C57BL/6J mice for at least ten generations), and their littermate controls Nppa+/+ and Npr1+/+ respectively were used. Mice were fed with a high fat diet (HFD) (60% kcal lipids) for 12 weeks and were housed in a pathogen-free barrier facility (12h light/dark cycle) with ad libitum access to water and food in standard animal care facility rooms at 21°C. Mice were weighted weekly and body composition was assessed by the use of an EchoMRI. At the end of the protocol, mice were killed and muscle tissues were rapidly excised and snap frozen in liquid nitrogen before being stored at -80°C. RNA seq libraries were constructed from these samples.