Cytochrome-c oxidase (CcO) is the terminal enzyme of the electron transport chain, where it catalyzes the reduction of oxygen to water. The oxygen-binding site is located near subunit I (CcO1), which is subject to tight regulatory control, including phosphorylation. Under diabetic conditions associated with inflammation, we observed increased phosphorylation at tyrosine 304 (Y304) of CcO1. This modification occurs specifically in dimeric CcO and certain respiratory supercomplexes, leading to reduced enzymatic activity. Our findings indicate that pY304 plays a key role in the diet- and strain-dependent regulation of mitochondrial function in diabetic heart tissue.