Previously, we showed that protein kinase B (Akt) activation increases intracellular ATP levels and decreases necrosis in renal proximal tubular cells (RPTC) injured by the nephrotoxicant, S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) (48). This study examined the role of Akt in improving mitochondrial function in DCVC-injured RPTC. Our data show a novel observation that phosphorylated (active) Akt is localized in mitochondria of non-injured RPTC, both in mitoplasts and the mitochondrial outer membrane. Mitochondrial levels of active Akt decreased in nephrotoxicant injured RPTC and this decrease was associated with mitochondrial dysfunction. DCVC decreased basal, uncoupled and state 3 respirations, ATP production, activities of complexes I, II and III, the mitochondrial membrane potential (_m), and F₀F₁-ATPase activity. Expressing constitutively active Akt in DCVC-injured RPTC increased the levels of phosphorylated Akt in mitochondria, reduced the decreases in basal and uncoupled respirations, increased complex I-coupled state 3 respiration and ATP production, enhanced activities of complex I, complex III and F₀F₁-ATPase , and improved _m. In contrast, inhibiting Akt activation by expressing dominant negative (inactive) Akt or using 20 µM LY294002 exacerbated decreases in electron transport rate, state 3 respiration and ATP production, _m and activities of complex I, complex III and F₀F₁-ATPase . In conclusion, our data show that Akt activation promotes mitochondrial respiration and ATP production in toxicant-injured RPTC by: 1) improving integrity of the respiratory chain and maintaining activities of complex I and complex III, 2) reducing decreases in _m, and 3) restoring F₀F₁-ATPase activity.