Hyperglycemia induces p38 MAPK-mediated renal proximal tubular cell (RPTC) apoptosis. The current study hypothesized that alteration of Akt signaling pathway by hyperglycemia may contribute to p38 MAPK activation and development of diabetic nephropathy (DN). Immunoblot analysis demonstrated a hyperglycemia-induced increase in Akt phosphorylation in diabetic kidneys at 1 month, peaking at 3 months, and dropping back to baseline by 6 months. Immunohistochemical staining with anti-pAkt antisera localized Akt phosphorylation to renal tubules. Maximal p38 MAPK phosphorylation was detected concomitant with increase in TUNEL positive cells and caspase-3 activity in 6 months diabetic kidneys. Exposure of cultured RPTCs to high glucose (HG; 22.5 mM) significantly increased Akt phosphorylation at 3, 6, and 9 hr, and decreased thereafter. In contrast, p38 MAPK phosphorylation was detected between 9 and 48 hr of HG treatment. Increased p38 MAPK activation at 24 and 48 hr coincided with increased apoptosis, demonstrated by increased caspase-3 activity at 24 hr and increased TUNEL positive cells at 48 hr of HG exposure. Blockade of p38 cascade with SB203850 inhibited HG-induced caspase-3 activation and TUNEL positive cells. Over-expression of constitutively active Akt abrogated HG-induced p38 MAPK phosphorylation and RPTC apoptosis. In addition, blockade of PI-3K/Akt pathway with LY294002 and silencing Akt expression with Akt siRNA, induced p38 MAPK phosphorylation in the absence of HG. These results collectively suggest that down-regulation of Akt activation during long term hyperglycemia contributes to enhanced p38 MAPK activation and RPTC apoptosis. Mechanism of down-regulation of Akt activation in 6 months STZ diabetic kidneys was attributed to decreased Akt-Hsp25, Akt-p38 interaction and decreased PTEN activity. Thus, PTEN or Hsp25 could serve as potential therapeutic targets to modulate Akt activation and control p38 MAPK-mediated diabetic complications.