The present study tested the hypotheses that overexpression of an intracellular Ang II (angiotensin II) fusion protein, mito-ECFP/Ang II, selectively in the mitochondria of mouse proximal tubule cells induces mitochondrial oxidative and glycolytic responses and elevates blood pressure via the Ang II/AT receptor/superoxide/NHE3 (the Na/H exchanger 3)-dependent mechanisms. A PT-selective, mitochondria-targeting adenoviral construct encoding Ad-sglt2-mito-ECFP/Ang II was used to test the hypotheses. The expression of mito-ECFP/Ang II was colocalized primarily with Mito-Tracker Red FM in mouse PT cells or with TMRM in kidney PTs. Mito-ECFP/Ang II markedly increased oxygen consumption rate as an index of mitochondrial oxidative response (69.5%; <0.01) and extracellular acidification rate as an index of mitochondrial glycolytic response (34%; <0.01). The mito-ECFP/Ang II-induced oxygen consumption rate and extracellular acidification rate responses were blocked by AT blocker losartan (<0.01) and a mitochondria-targeting superoxide scavenger mito-TEMPO (<0.01). By contrast, the nonselective NO inhibitor L-NAME alone increased, whereas the mitochondria-targeting expression of AT receptors (mito-AT/GFP) attenuated the effects of mito-ECFP/Ang II (<0.01). In the kidney, overexpression of mito-ECFP/Ang II in the mitochondria of the PTs increased systolic blood pressure 12±3 mm Hg (<0.01), and the response was attenuated in PT-specific PT- and PT- mice (<0.01). Conversely, overexpression of AT receptors selectively in the mitochondria of the PTs induced natriuretic responses in PT- and PT- mice (<0.01). Taken together, these results provide new evidence for a physiological role of PT mitochondrial Ang II/AT/superoxide/NHE3 and Ang II/AT/NO/NHE3 signaling pathways in maintaining blood pressure homeostasis.